Cell membrane penetrating conjugates

ABSTRACT

A cell penetrating conjugate comprising a recombinant β helical protein linked to a functional molecule wherein the β helical protein length is in the range of from 5 nm to 25 nm, suitably, from 10 nm to 15 nm and width is in the range of from 1 nm to 5 nm, suitably, from 1 nm to 3 nm. Processes for preparing said conjugates and uses thereof are also disclosed.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML file format and is hereby incorporatedby reference in its entirety. Said XML copy, created on Feb. 6, 2023, isnamed 51483-002002_SL.xml and is 36,402 bytes in size.

FIELD OF INVENTION

The present disclosure broadly relates to the field of drug delivery tothe cytoplasm of cells and particularly discloses a conjugate comprisinga recombinant protein linked to a functional molecule for penetratingcellular membranes, methods of preparing said conjugates and usesthereof.

BACKGROUND OF THE INVENTION

A cell membrane is a semi-permeable membrane which separates the innerenvironment of a cell from its external environment. Membranes ofprokaryotic and eukaryotic cells while differing in some properties andcomposition both comprise a semi-permeable bilayer structure ofphospholipids. The semi-permeable nature of the cell membrane makes itselective for the type of molecules able to penetrate it. Thosemolecules capable of penetrating cell membranes hold promise for use incell labelling, cell penetration, cell delivery, drug uptake, genetherapy and many other applications which involve penetration of thecell membrane.

There are certain peptides which can penetrate the cell membrane andtranslocate to the cytosol, such peptides are termed as cell penetratingpeptides (CPPs). CPP conjugates, where the CPP is attached to one ormore functional molecules, have been studied as a means to transportvarious biologically active molecules across the membrane. For example,absorption of insulin was drastically increased (6-8 times) in Caco-2cells when treated with the CPP conjugate, CPP-insulin (Liang et al.,Biochem. Biophys. Res. Commun.; 2005; 335(3): 734-738). Similar resultswere seen for a conjugate comprising the Tat peptide (ibid.). Anotherstudy has reported use of a short amphipathic peptide carrier Pep-1which could penetrate the membrane and deliver various peptides andproteins in several cell lines (Morris et al., Nature Biotechnol., 2001,1173-1176).

CPPs have also been used to study the efficient delivery of variousanti-cancer drugs as drug-CPP conjugates which could penetrate cellmembranes more efficiently than the drug alone because of the propertiesof the CPP. One such study reported the use of Tat protein conjugatedwith a CK2 inhibitor (P15) to treat solid tumours (Perea et al., CancerRes. 2004, 7127-7129).

There are also several molecular transporters which can delivermolecules across cell membranes. Guanidinium-rich molecular transporters(GR-MoTrs) comprising peptide and non-peptide agents have been shown topenetrate cell membrane owing to their number and spatial array ofguanidinium groups. GR-MoTrs can enhance delivery of various cargosincluding small molecules, metals, imaging agents, iron particles, andproteins inside mammalian cells (Wender et al., Adv. Drug Deliv. Rev.2008, 452-472; Wender et al., Drug Discov. Today Technol. 2012,e49-e55).

US20130137644 discloses a conjugate made up of a cell penetratingpeptide, nucleic acid and a hydrophilic polymer which can penetrate cellmembranes with increased efficiency. The nucleic acid used in theconjugate is described as preferably being an siRNA, with polyethyleneglycol (PEG) as the hydrophilic polymer.

US20040176282 discloses methods and uses of compositions for thecellular delivery of nucleic acids, polypeptides, fluorophores,molecular complexes. The intracellular release of the biologicallyactive molecules after cell penetration is stimulated by light-activateddispersal of the complex. This system helps in repressing the biologicalfunctions of the molecules while being a part of the complex, but onceinside the cell and upon light activation it can be dispersed and itsbiological activity can be restored.

One example of a promising and widely used technique involvingfunctional molecules crossing the cell membrane barrier is gene therapy.Gene therapy involves delivering a gene of interest to cells tocompensate for abnormal activity of genes or to provide a beneficialprotein. Gene therapy has proven beneficial in treating diseases likechronic lymphocytic leukaemia, X-linked SCID, multiple myeloma,haemophilia amongst many others. Many life-threatening diseases have anunderlying genetic origin, i.e. the diseases are due to malfunctioningor lack of proper functioning displayed by one or more associated genes.Gene therapy has shown promise for treatment of such disease conditions.However, gene therapy still faces a challenge in terms of delivering therequired genes across a cell membrane. To date, two approaches fordelivering the genes have been used—viral based and non-viral based.

The viral based approach for gene therapy makes use of attenuatedviruses as vectors in which the desired gene is cloned and transferredinto the required cells via a process known as transduction. Thisapproach, has the advantage of suitable integration of the deliveredgene into the genome of the cells but suffers from other disadvantages,one of them being a tendency to induce cancer in case of inappropriategenome integration. The non-viral based approach includes the use ofinjecting isolated DNA into the cells and the use of cationic lipids forsurrounding a plasmid DNA (lipofection). The non-viral approaches do notrequire any integration of the gene into the genome and are inefficientin transferring the required genes to other cells in the tissues.Therefore gene therapy, although a promising and an excellent techniquefor treating a number of life-threatening disorders, suffers from theproblem of delivery of genes into the cells.

For the most common inherited disorders, such as cystic fibrosis ormuscular dystrophy, effective gene therapy is likely to remain achallenge due to the difficulties in delivering the genetic materialinto the cell. There is not yet a simple way to deliver genes to asignificant proportion of cells in tissues such as the lung epitheliumor skeletal muscle (Collins et al., Proc. R. Soc. B. Vol. 282. No. 1821.The Royal Society, 2015). Hence, an effective mechanism of cell deliveryis required which can greatly enhance the benefits of gene therapy andexpand the avenues for providing promising treatments for manylife-threatening diseases.

In addition, and general to the delivery of all functional molecules tothe cells, is that most of the mechanisms developed for cellulardelivery suffer rely on an endocytosis-dependent mechanism for gainingentry inside the cells. The efficiency of translocation is a major areaof concern in endocytosis dependent pathways due to, for example, thepossibility of drugs getting trapped in endosomes or degraded inlysosomes. Hence there is a pressing need for devising novel mechanismsfor penetrating cell membranes, which can be more reliable andefficacious.

SUMMARY OF INVENTION

These and other features, aspects, and advantages of the present subjectmatter will be better understood with reference to the followingdescription and appended claims. This summary is provided to introduce aselection of concepts in a simplified form. This summary is not intendedto identify key features or essential features of the claimed subjectmatter, nor is it intended to be used to limit the scope of the claimedsubject matter.

In a first aspect of the present disclosure, there is provided acell-penetrating conjugate comprising at least one recombinant β helicalprotein molecule linked to a functional molecule, wherein the β helicalprotein length is in the range of 5 nm-25 nm, suitably, 10 nm-15 nm andwidth is in the range of 1 nm-5 nm, suitably 1 nm-3 nm.

In a second aspect of the present disclosure, there is provided aprocess for transferring a functional molecule into a cell, said processcomprising: (a) linking the functional molecule to a recombinant βhelical protein to obtain a conjugate; and (b) contacting thecell-penetrating conjugate with at least one cell; wherein contactingthe conjugate with the at least one cell transfers the nucleic acidmolecule into the cell, and wherein the β helical protein length is inthe range of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the rangeof 1 nm-5 nm, suitably 1 nm-3 nm.

In a third aspect, the present disclosure is directed to use of the cellpenetrating conjugate of the first aspect of the invention fordelivering a functional molecule into cells, wherein the functionalmolecule is selected from the group consisting of dyes, drugs, metal,drug-metal, proteins, enzymes, antibodies, nucleic acids,polysaccharides, nuclear localising signals, nanoparticles andcombinations thereof.

In a fourth aspect, the present disclosure is directed to use of thecell penetrating conjugate of the first aspect of the invention for cellpenetration.

In a fifth aspect, the present disclosure is directed to use of the cellpenetrating conjugate of the first aspect of the invention for celllabelling.

In a sixth aspect of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm.

In a seventh aspect of the present disclosure, there is provided aprocess for transferring a nucleic acid molecule into a cell, saidprocess comprising: (i) linking the nucleic acid molecule to at leastone recombinant β helical protein via at least one linker to obtain aconjugate; and (ii) contacting the conjugate with at least one cell,wherein contacting the conjugate with the at least one cell transfersthe nucleic acid molecule into the cell, and wherein the recombinant βhelical protein length is in the range of 5 nm-25 nm, suitably, 10 nm-15nm and width is in the range of 1 nm-5 nm, suitably 1 nm-3 nm.

In an eighth aspect, the present disclosure is directed to use of thecell penetrating conjugate of the sixth aspect of the invention as atransfecting agent.

In a ninth aspect, the present disclosure is directed to use of the cellpenetrating conjugate of the sixth aspect of the invention for genetherapy.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

The following drawings form a part of the present specification and areincluded to further illustrate aspects of the present disclosure. Thedisclosure may be better understood by reference to the drawings incombination with the detailed description of the specific embodimentspresented herein.

FIG. 1 shows the CD (circular dichroism) spectra of the isolated A1bGprotein, in accordance with the present disclosure.

FIG. 2 shows an agarose gel containing purified plasmids (containingA1bG and EfsQNR genes), in accordance with the present disclosure.

FIG. 3 shows a poly-acrylamide gel containing purified proteins (A1bGand EfsQNR), in accordance with the present disclosure.

FIG. 4 shows MALDI-TOF mass spectral analysis of the EfsQNR and A1bGproteins.

FIG. 5 shows the graphical representation of characterization oflabelled proteins (A1bG-NHSC and EfsQNR-NHSC) by UV-visiblespectrophotometry, in accordance with an embodiment of the presentdisclosure.

FIGS. 6A-6D show differential labelling of Hela cells by labelledproteins (conjugates) A1bG-NHSC (FIG. 6C), EfsQNR-NHSC (FIG. 6D) andTtCuA-NHSC (FIG. 6B), relative to a control (FIG. 6A), in accordancewith embodiments of the present disclosure.

FIGS. 7A and 7B show labelling of Hela cells by EfsQNR labelled withATTO-520 (commercially available green fluorescent dye), in accordancewith an embodiment of the present disclosure.

FIG. 8 shows labelling of Hela cells by EfsQNR labelled with ATTO-390(commercially available blue fluorescent dye), in accordance with anembodiment of the present disclosure.

FIGS. 9A and 9B show labelling of microglial cells by EfsQNR labelledwith ATTO-520 (commercially available green fluorescent dye), inaccordance with an embodiment of the present disclosure (FIG. 9A); anddifferential labelling of microglial cells by EfsQNR labelled withATTO-520 (FIG. 9B).

FIG. 10 shows labelling of keratinocyte cells by EfsQNR labelled withATTO-520 (commercially available green fluorescent dye), in accordancewith an embodiment of the present disclosure (Panel A); and differentiallabelling of keratinocyte cells by EfsQNR labelled with ATTO-520 (PanelB).

FIG. 11 shows labelling of SH-SYSY cells by EfsQNR labelled withATTO-520 (commercially available green fluorescent dye), in accordancewith an embodiment of the present disclosure.

FIG. 12 shows labelling of mouse ES cells by EfsQNR labelled withATTO-520 (commercially available green fluorescent dye), in accordancewith an embodiment of the present disclosure.

FIG. 13 shows labelling of E. coli cells by EfsQNR labelled withATTO-520 (commercially available green fluorescent dye), in accordancewith an embodiment of the present disclosure.

FIG. 14 shows labelling of yeast (kluveromyces) cells by EfsQNR labelledwith ATTO-520 (commercially available green fluorescent dye), inaccordance with an embodiment of the present disclosure.

FIGS. 15A-15D show the results of standard FACS sorting of HeLa cellstreated with a conjugate labelled with the conjugate EfsQNR-ATTO-647Nfor 10 mins (FIG. 15B), 1 h (FIG. 15C) and 3 h (FIG. 15D) compared tountreated cells (FIG. 15A).

FIG. 16 shows percentage cellular uptake of a drug as part of aconjugate in accordance with an embodiment of the present disclosure, ascompared to unconjugated drug.

FIG. 17 shows percentage viability of cells (Hela and HepG2) aftertreatment with Cisplatin® a chemotherapy drug as part of a conjugatewith the protein EfsQNR (labelled CYDD) in accordance with an embodimentof the present disclosure, as compared to unconjugated drug.

FIG. 18 shows a plasmid vector carrying mcherry gene, in accordance withan embodiment of the present disclosure.

FIG. 19 shows a process for preparing a conjugate, in accordance with anembodiment of the present disclosure.

FIG. 20 shows a representation of transfection using a conjugate, inaccordance with an embodiment of the present disclosure.

FIG. 21 shows confocal microscopy images of HeLa cells aftertransfection with a conjugate in accordance with the present invention;the conjugate comprising the mcherry gene coding for RFP (RedFluorescent Protein) linked via copper [II] phenanthroline to the EfsQNRprotein.

FIG. 22 shows real-time direct penetration of a cell membrane by anEfsQNR-ATTO 520 conjugate in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Those skilled in the art will be aware that the present disclosure issubject to variations and modifications other than those specificallydescribed. It is to be understood that the present disclosure includesall such variations and modifications. The disclosure also includes allsuch steps, features, compositions, and compounds referred to orindicated in this specification, individually or collectively, and anyand all combinations of any or more of such steps or features.

Definitions

For convenience, before further description of the present disclosure,certain terms employed in the specification, and examples are delineatedhere. These definitions should be read in the light of the remainder ofthe disclosure and understood as by a person of skill in the art. Theterms used herein have the meanings recognized and known to those ofskill in the art, however, for convenience and completeness, particularterms and their meanings are set forth below.

Unless otherwise indicated, the practice of the present inventionemploys conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA technology, and chemical methods, whichare within the capabilities of a person of ordinary skill in the art.Such techniques are also explained in the literature, for example, M. R.Green, J. Sambrook, 2012, Molecular Cloning: A Laboratory Manual, FourthEdition, Books 1-3, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Ausubel, F. M. et al. (1995 and periodic supplements;Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley &Sons, New York, N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNAIsolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M.Polak and James O′D. McGee, 1990, In Situ Hybridisation: Principles andPractice, Oxford University Press; M. J. Gait (Editor), 1984,Oligonucleotide Synthesis: A Practical Approach, IRL Press; and D. M. J.Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA StructurePart A: Synthesis and Physical Analysis of DNA Methods in Enzymology,Academic Press. Each of these general texts is herein incorporated byreference.

The articles ‘a’, ‘an’ and ‘the’ are used to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle.

As used herein, the term ‘comprising’ means any of the recited elementsare necessarily included and other elements may optionally be includedas well. ‘Consisting essentially of’ means any recited elements arenecessarily included, elements which would materially affect the basicand novel characteristics of the listed elements are excluded, and otherelements may optionally be included. ‘Consisting of’ means that allelements other than those listed are excluded. Embodiments defined byeach of these terms are within the scope of this invention.

The term ‘including’ is used to mean ‘including but not limited to’.‘Including’ and ‘including but not limited to’ are used interchangeably.

As used herein, the term ‘cell membrane’ is a biological membrane whichis present in both prokaryotic and eukaryotic cells and separates theinner and outer environment of a cell. It acts as a semi-permeablebarrier which checks the transport of substances in and out of the celland is typically formed of a phospholipid bilayer. The membrane acts asa support and helps in maintaining the shape and structure of a cell.

As used herein the term ‘β helical protein’ means a protein forming a βhelical secondary structure. β helical proteins are formed from agenerally parallel association between adjacent β strands of a peptidechain. A β helical protein can be a right handed β helical or a lefthanded β helical depending on the direction of coiling of the helixstructure.

As used herein the term ‘Pentapeptide-repeat protein (PRP)’ means βhelical proteins consisting of a tandemly repeated pentapeptide. Inembodiments, the tandemly repeated pentapeptide has the consensussequence (STAV)₁(DN)₂(LF)₃(STR)₄(G)₅ (SEQ ID NO: 18). The PRP family haswell over 500 members in the prokaryotic and eukaryotic kingdom.

As used herein the term ‘functional molecule’ is any molecule that has autility within a cell. Examples of functional molecules suitable for usein the present invention include dyes, drug molecules, proteins,enzymes, antibodies, and nucleic acids.

As used herein the term ‘cell penetrating peptide’ or ‘CPP’ relates topeptides sequences that facilitate cellular intake/uptake of variousfunctional molecules. Cell penetrating peptides generally deliver thefunctional molecule directly through the cell membrane avoiding the needfor endocytosis mediated pathways for cellular entry.

As used herein the term ‘P-cadherin’ refers to a cell to cell adhesionmolecule having a homeostatic function in normal tissues. The overexpression of this molecule is associated with significant tumourpromoting effects in the breast, ovarian, prostate, endometrial, skin,gastric, pancreas and colon neoplasms.

As used herein the term ‘NHS-coumarin’ or ‘NHSC’ refers to a fluorescentdye widely used in cell biology techniques. It is a common name for7-(diethylamino)coumarin-3-carboxylic acid N-succinimidyl ester having amolecular weight of 358.35 g/mol. NHS-coumarin (NHSC) has an excitationwavelength of 445 nm and an emission wavelength at 482 nm. Onobservation under fluorescent microscopy, it emits green fluorescenceindicating the location and quantification of the molecule conjugatedalong with this dye.

As used herein the term ‘phosphatidyl choline’ defines a class ofphospholipids molecules that incorporate a choline as a headgroup.Phosphatidyl choline may be used as a signalling molecule thatfacilitates selective binding and attachment with cell membranes.

As used herein the term ‘Hoechst 33342’ refers to a solution offluorescent dye that is used for both fixed and live cell staining ofDNA and nuclei in cellular imaging techniques. Hoechst 33342 is acell-permeable DNA stain having an excitation wavelength of 460 nm andan emission wavelength of 490 nm and it preferentially binds toadenine(A)-thymine(T) region of DNA.

As used herein the term ‘ATTO 520’, ‘ATTO 390’ and ‘ATTO 647N’ referrespectively to fluorescent dyes developed by the ATTO-Tec GmbH andcommercially available from Sigma Aldrich.

As used herein the term ‘Ruthenium metal complex’ refers to thecoordination complex of ruthenium metal which is known to possessanti-cancer activities. Octahedral ruthenium (III) and ruthenium (II)complexes display anti-neoplastic activities on many experimentaltumours. Ruthenium metal complex is considered as an excellentalternative to circumvent the side-effects of platinum based compounds.A non-limiting example of a ruthenium metal complex that has beendemonstrated for use in the present invention is tricarbonyl dichlororuthenium(II) (ex-Sigma Aldrich).

The term ‘nucleic acid’ as used herein, is a single or double strandedcovalently-linked sequence of nucleotides in which the 3′ and 5′ ends oneach nucleotide are joined by phosphodiester bonds. The polynucleotidemay be made up of deoxyribonucleotide bases or ribonucleotide bases.Nucleic acids may include DNA and RNA, and are typically manufacturedsynthetically, but may also be isolated from natural sources. Nucleicacids may further include modified DNA or RNA, for example DNA or RNAthat has been methylated or that has been subject to chemicalmodification, for example 5′-capping with 7-methylguanosine,3′-processing such as cleavage and polyadenylation, and splicing, orlabelling with fluorophores or other compounds. Nucleic acids may alsoinclude synthetic nucleic acids (XNA), such as hexitol nucleic acid(HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA),glycerol nucleic acid (GNA), locked nucleic acid (LNA) and peptidenucleic acid (PNA). Hence, where the terms ‘DNA’ and ‘RNA’ are usedherein it should be understood that these terms are not limited to onlyinclude naturally occurring nucleotides. Sizes of nucleic acids, alsoreferred to herein as ‘polynucleotides’ are typically expressed as thenumber of base pairs (bp) for double stranded polynucleotides, or in thecase of single stranded polynucleotides as the number of nucleotides(nt). One thousand bp or nt equal a kilobase (kb). Polynucleotides ofless than around 100 nucleotides in length are typically called‘oligonucleotides’.

As used herein, the terms ‘3″ (3 prime’) and ‘5′’ (‘5 prime’) take theirusual meanings in the art, i.e. to distinguish the ends ofpolynucleotides. A polynucleotide has a 5′ and a 3′ end andpolynucleotide sequences are conventionally written in a 5′ to 3′direction.

The term ‘amino acid’ in the context of the present invention is used inits broadest sense and is meant to include naturally occurring L α-aminoacids or residues. The commonly used one and three letter abbreviationsfor naturally occurring amino acids are used herein: A=Ala; C=Cys;D=Asp; E=Glu; F=Phe; G=Gly; H=His; I=Ile; K=Lys; L=Leu; M=Met; N=Asn;P=Pro; Q=Gln; R=Arg; S=Ser; T=Thr; V=Val; W=Trp; and Y=Tyr (Lehninger,A. L., (1975) Biochemistry, 2d ed., pp. 71-92, Worth Publishers, NewYork). The general term ‘amino acid’ further includes D-amino acids,retro-inverso amino acids as well as chemically modified amino acidssuch as amino acid analogues, naturally occurring amino acids that arenot usually incorporated into proteins such as norleucine, andchemically synthesised compounds having properties known in the art tobe characteristic of an amino acid, such as β-amino acids. For example,analogues or mimetics of phenylalanine or proline, which allow the sameconformational restriction of the peptide compounds as do natural Phe orPro, are included within the definition of amino acid. Such analoguesand mimetics are referred to herein as ‘functional equivalents’ of therespective amino acid. Other examples of amino acids are listed byRoberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Grossand Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., N.Y. 1983,which is incorporated herein by reference.

A ‘polypeptide’ is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or in vitro by synthetic means.Polypeptides of less than around 12 amino acid residues in length aretypically referred to as ‘peptides’ and those between about 12 and about30 amino acid residues in length may be referred to as ‘oligopeptides’.The term ‘polypeptide’ as used herein denotes the product of a naturallyoccurring polypeptide, precursor form or proprotein. Polypeptides canalso undergo maturation or post-translational modification processesthat may include, but are not limited to: glycosylation, proteolyticcleavage, lipidization, signal peptide cleavage, propeptide cleavage,phosphorylation, and such like. The term ‘protein’ is used herein torefer to a macromolecule comprising one or more polypeptide chains.

The term ‘isolated’, when applied to a polynucleotide or proteinsequence, denotes that the sequence has been removed from its naturalorganism of origin and is, thus, free of extraneous or unwanted codingor regulatory sequences. The isolated sequence is suitable for use inassembly of compositions and nanostructures of the present invention.Such isolated sequences may include cDNAs and RNAs.

According to the present invention, homology to the nucleic acid orprotein sequences described herein is not limited simply to 100%sequence identity. Any closely related nucleic acid or protein sequencesto those specified herein that demonstrate functional and/or biochemicalequivalence are considered within the scope of the present invention asdefined by the claims.

The term ‘signal sequence’ in the context of the present invention meansnuclear localizing sequence or recognition sequences for different cellorganelles or nucleic acid binding domains such as zinc finger bindingproteins.

As used herein the term ‘carrier’ means substances that serve asmechanisms to improve the delivery and the effectiveness of the drug.

As used herein the term ‘diluent’ (also referred to as filler, dilutant,or thinner) means a diluting agent.

As used herein the term ‘excipient’ refers to an inactive substance thatserves as a vehicle or medium for a drug or other active substance.Excipients include colouring agents, humectants, preservatives,emollients and combinations thereof.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the disclosure, the preferred methods, andmaterials are now described. All publications mentioned herein areincorporated herein by reference.

Sequences SEQ ID NO: 1 depicts amino acid sequence of A1bG protein.MPAKTLESKDYCGESFVSEDRSGQSLESIRFEDCT FRQCNFTEAELNRCKFRECEFVDCNLSLISIPQTSFMEVRFVDCKMLGVNWTSAQWPSVKMEGALSFERC ILNDSLFYGLYLAGVKMVECRIHDANFTEADCEDADFTQSDLKGSTFHNTKLTGASFIDAVNYHIDIFHN DIKRARFSLPEAASLLNSLDIELSDSEQ ID NO: 2 depicts amino acid sequence of EfsQNR protein.GSHMKITYPLPPNLPEQLPLLTNCQLEDEAILENH LYQQIDLPNQEVRNLVFRDAVFDHLSLANGQFASFDCSNVRFEACDFSNVEWLSGSFHRVTFLRCNLTGT NFADSYLKDCLFEDCKADYASFRFANFNLVHFNQTRLVESEFFEVTWKKLLLEACDLTESNWLNTSLKGL DFSQNTFERLTFSPNYLSGLKVTPEQAIYLASALGLVIT SEQ ID NO: 3 depicts amino acidsequence of anti-freeze protein from Tenebrio molitor.QCTGGADCTSCTGACTGCGNCPNAVTCTNSQHCVK ANTCTGSTDCNTAQTCTNSKDCFEANTCTDSTNCYKATACTNSSGCPGH SEQ ID NO: 4 depicts amino acidsequence of anti-freeze protein from Rhagium inquisitor.GYSCRAVGVDGRAVTDIQGTCHAKATGAGAMASGT SEPGSTSTATATGRGATARSTSTGRGTATTTATGTASATSNAIGQGTATTTATGSAGGRATGSATTSSSA SQPTQTQTITGPGFQTAKSFARNTATTTVTASHHHHHH SEQ ID NO: 5 depicts amino acid sequence of anti-freeze proteinfrom Spruce Budworm (Choristoneura fumiferana).DGSCTNTNSQLSANSKCEKSTLTNCYVDKSEVYGT TCTGSRFDGVTITTSTSTGSRISGPGCKISTCIITGGVPAPSAACKISGCTFSAN SEQ ID NO: 6 depicts amino acidsequence of QNRB1 protein. GSHMALALVGEKIDRNRFTGEKIENSTFFNCDFSGADLSGTEFIGCQFYDRESQKGCNFSRAMLKDAI FKSCDLSMADFRNSSALGIEIRHCRAQGADFRGASFMNMITTRTWFCSAYITNTNLSYANFSKVVLEK CELWENRWIGAQVLGATFSGSDLSGGEFSTFDWRAANFTHCDLTNSELGDLDIRGVDLQGVKLDNYQASL LMERLGIAVIGSEQ ID NO: 7 depicts amino acid sequence of UDP-N-acetylglucosamineacyltransferase protein. MIDKSAFVHPTAIVEEGASIGANAHIGPFCIVGPHVEIGEGTVLKSHVVVNGHTKIGRDNEIYQFASIGE VNQDLKYAGEPTRVEIGDRNRIRESVTIHRGTVQGGGLTKVGSDNLLMINAHIAHDCTVGNRCILANNAT LAGHVSVDDFAIIGGMTAVHQFCIIGAHVMVGGCSGVAQDVPPYVIAQGNHATPFGVNIEGLKRRGFSRE AITAIRNAYKLIYRSGKTLDEVKPEIAELAETYPEVKAFTDFFARSTRGLIR SEQ ID NO: 8 depicts amino acidsequence of NP275 protein from Nostoc punctiforme.MGSSHHHHHHSSGLVPRGSHMDVEKLRQLYAAG ERDFSIVDLRGAVLENINLSGAILHGAMLDEANLQQANLSRADLSGATLNGADLRGANLSKADLSDAIL DNAILEGAILDEAVLNQANLKAANLEQAILSHANIREADLSEANLEAADLSGADLAIADLHQANLHQAA LERANLTGANLEDANLEGTILEGGNNNLATSEQ ID NO: 9 depicts amino acid sequence of pectate lyase C.ATDTGGYAATAGGNVTGAVSKTATSMQDIVNIIDA ARLDANGKKVKGGAYPLVITYTGNEDSLINAAAANICGQWSKDPRGVEIKEFTKGITIIGANGSSANFGI WIKKSSDVVVQNMRIGYLPGGAKDGDMIRVDDSPNVWVDHNELFAANHECDGTPDNDTTFESAVDIKGA SNTVTVSYNYIHGVKKVGLDGSSSSDTGRNITYHHNYYNDVNARLPLQRGGLVHAYNNLYTN ITGSGLNVRQNGQALIENNWFEKAINPVTSRYDGKNFGTWVLKGNNITKPADFSTYSITWTADTKPYVNA DSWTSTGTFPTVAYNYSPVSAQCVKDKLPGYAGVGKNLATLTSTACK SEQ ID NO: 10 depicts amino acidsequence of pectate lyase from Caldicellulosiruptor besciiVGTNTGGVLVITDTIIVKSGQTYDGKGIKIIAQGM GDGSQSENQKPIFKLEKGANLKNVIIGAPGCDGIHCYGDNVVENVVWEDVGEDALTVKSEGVVEVIGGSA KEAADKVFQLNAPCTFKVKNFTATNIGKLVRQNGNTTFKVVIYLEDVTLNNVKSC VAKSDSPVSELWYH NLNVNNCKTLFEFPSQSQIHQYSEQ ID NO: 11 depicts amino acid sequence of carbonic anhydrase fromMethanosarcina thermophila. QEITVDEFSNIRENPVTPWNPEPSAPVIDPTAYIDPQASVIGEVTIGANVMVSPMASIRSDEGM PIFVGDRSNVQDGVVLHALETINEEGEPIEDNIVEVDGKEYAVYIGNNVSLAHQSQVHGPAAVGDDTFIG MQAFVFKSKVGNNCVLEPRSAAIGVTIPDGRYIPAGMVVTSQAEADKLPEVTDDYAYSHTNEAVVYVNVH LAEGYKETSSEQ ID NO: 12 depicts amino acid sequence of Pectin lyase A protein fromAspergillus niger. VGVSGSAEGFAKGVTGGGSATPVYPDTIDELVSYLGDDEARVIVLTKTFDFTDSEGTTTGTGCAPWGTAS ACQVAIDQDDWCENYEPDAPSVSVEYYNAGTLGITVTSNKSLIGEGSSGAIKGKGLRIVSGAENIIIQNI AVTDINPKYVWGGDAITLDDCDLVWIDHVTTARIGRQHYVLGTSADNRVSLTNNYIDGVSDYSATCDGYH YWAIYLDGDADLVTMKGNYIYHTSGRSPKVQDNTLLHAVNNYWYDISGHAFEIGEGGYVLAEGNVFQNVD TVLETYEGEAFTVPSSTAGEVCSTYLGRDCVINGFGSSGT FSEDSTSFLSDFEGKNIASASAYTSVASR VVANAGQGNLSEQ ID NO: 13 depicts amino acid sequence of TtCuA protein.AYTLATHTAGVIPAGKLERVDPTTVRQEGPWADPA QAVVQTGPNQYTVYVLAFAFGYQPNPIEVPQGAEIVFKITSPDVIHGFHVEGTNINVEVLPGEVSTVRYT FKRPGEYRIICNQYCGLGHQNMFGTIVVKESEQ ID NO: 14 depicts a signal sequencefor targeting the nucleus of the cell. PAAKRVKCDSEQ ID NO: 15 depicts a signal sequence for targeting the endoplasmicreticulum of the cell. YPYDVPDYAKDELSEQ ID NO: 16 depicts a signal sequencefor targeting the mitochondria of the cell. MLSLRQSIRFFKPATRTLCSSRYLLSEQ ID NO: 17 depicts a signal sequencefor targeting the P-cadherin-over expressing breast cancer cells.LSTAADMQGVVTDGMASGLDKDYLKPDD SEQ ID NO: 18 depicts a consensussequence in a pentapeptide-repeat protein. (STAV)₁(DN)₂(LF)₃(STR)₄(G)₅SEQ ID NO: 19 depicts a nucleic acid sequence otA1bG gene.ATGCCGGCGAAAACCCTGGAAAGCAAAGATTATTG CGGCGAAAGCTTTGTGAGCGAAGATCGCAGCGGCCAGAGCCTGGAAAGCATTCGCTTTGAAGATTGCACC TTTCGCCAGTGCAACTTTACCGAAGCGGAACTGAACCGCTGCAAATTTCGCGAATGCGAATTTGTGGATT GCAACCTGAGCCTGATTAGCATTCCGCAGACCAGCTTTATGGAAGTGCGCTTTGTGGATTGCAAAATGCT GGGCGTGAACTGGACCAGCGCGCAGGCGGGCGCGCTGAGCTTTGAACGCTGCATTCTGAACGATAGCCTG TTTTATGGCCTGTATCTGGCGGGCGTGAAAATGGTGGAATGCCGCATTCATGATGCGAACTTTACCGAAG CGGATTGCGAAGATGCGGATTTTACCCAGAGCGATCTGAAAGGCAGCACCTTTCATAACACCAAACTGAC CGGCGCGAGCTTTATTGATGCGGTGAACTATCATATTGATATTTTTCATAACGATATTAAACGCGCGCGC TTTAGCCTGCCGGAAGCGGCGAGCCTGCTGAACAGCCTGGATATTGAACTGAGCGAT SEQ ID NO: 20 depicts a nucleic acidsequence of EfsQNR gene. GGCAGCCATATGAAAATTACCTATCCGCTGCCGCCGAACCTGCCGGAACAGCTGCCGCTGCTGACCAACT GCCAGCTGGAAGATGAAGCGATTCTGGAAAACCATCTGTATCAGCAGATTGATCTGCCGAACCAGGAAGT GCGCAACCTGGTGTTTCGCGATGCGGTGTTTGATCATCTGAGCCTGGCGAACGGCCAGTTTGCGAGCTTT GATTGCAGCAACGTGCGCTTTGAAGCGTGCGATTTTAGCAACGTGGAATGGCTGAGCGGCAGCTTTCATC GCGTGACCTTTCTGCGCTGCAACCTGACCGGCACCAACTTTGCGGATAGCTATCTGAAAGATTGCCTGTT TGAAGATTGCAAAGCGGATTATGCGAGCTTTCGCTTTGCGAACTTTAACCTGGTGCATTTTAACCAGACC CGCCTGGTGGAAAGCGAATTTTTTGAAGTGACCTGGAAAAAACTGCTGCTGGAAGCGTGCGATCTGACCG AAAGCAACTGGCTGAACACCAGCCTGAAAGGCCTGGATTTTAGCCAGAACACCTTTGAACGCCTGACCTT TAGCCCGAACTATCTGAGCGGCCTGAAAGTGACCCCGGAACAGGCGATTTATCTGGCGAGCGCGCTGGGC CTGGTGATTACCSEQ ID NO: 21 depicts a nucleic acid sequence of TtCuA geneGCGTATACCCTGGCGACCCATACCGCGGGCGTGAT TCCGGCGGGCAAACTGGAACGCGTGGATCCGACCACCGTGCGCCAGGAAGGCCCGTGGGCGGATCCGGCG CAGGCGGTGGTGCAGACCGGCCCGAACCAGTATACCGTGTATGTGCTGGCGTTTGCGTTTGGCTATCAGC CGAACCCGATTGAAGTGCCGCAGGGCGCGGAAATTGTGTTTAAAATTACCAGCCCGGATGTGATTCATGG CTTTCATGTGGAAGGCACCAACATTAACGTGGAAGTGCTGCCGGGCGAAGTGAGCACCGTGCGCTATACC TTTAAACGCCCGGGCGAATATCGCATTATTTGCAACCAGTATTGCGGCCTGGGCCATCAGAACATGTTTG GCACCATTGTGGTGAAAGAASEQ ID NO: 22 depicts a signal sequencefor targeting to actin in the cell. GDVQKKRWLFETKPLDSEQ ID NO: 23 depicts a signal sequencefor targeting to tubulin in the cells. VQSKCGSKDNIKHVPGGGSEQ ID NO. 24: depicts an amino acid sequence of a zinc finger proteinMERPYACPVESCDRRFSDSSNLTRHIRIHTGQKPFQCRICMRNFSRSDHLTTHIRTHTGEKPFACDICGRKF ARSDERKRHTKIHLRQKDSEQ ID NO. 25: depicts a nucleic acid sequence of mcherry gene.GTGAGCAAGGGCGAGGAGGATAACATGGCCATCAT CAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGC GAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGC CCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGA CATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGAC GGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTCCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGC GCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGA GCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGC CACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACG TCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGA GGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAGTAATCTAGAGGGCCCTATTCTATAGTG TCACC.SEQ ID NO. 26: depicts a nucleic acid sequence of a primer for PCRamplification of the A1bG gene. ATCCCGCTCATATGCCGGCCAAGACCCTTGSEQ ID NO. 27: depicts a nucleic acid sequence of a primer for PCRamplification of the A1bG gene. ATCCCGCTCTCGAGTCAATCGGACAGCTCGATATCSEQ ID NO. 28: depicts a nucleic acid sequence of a primer for PCRamplification of the EfsQNR gene. ATCCCGCTCATATGAAAATAACTTATCCCTTGCCASEQ ID NO. 29: depicts a nucleic acid sequence of a primer for PCRamplification of the EfsQNR gene. ATCCCGCTCTCGAGTTAGGTAATCACCAAACCAAGT

The present invention also provides for conjugates comprising arecombinant protein and a functional molecule for penetrating cellularmembranes, and uses thereof that have substantially similar sequenceidentity or homology to that of SEQ ID NOs: 1 to 12. The term‘substantially similar sequence identity’ is used herein to denote alevel of sequence similarity of from about 50%, 60%, 70%, 80%, 90%, 95%to about 99% identity. Percent sequence identity can be determined usingconventional methods, for example those described in Henikoff andHenikoff Proc. Natl. Acad. Sci. USA 1992; 89:10915, and Altschul et al.Nucleic Acids Res. 1997; 25:3389-3402 for nucleic acids; and forproteins via comparison after alignment using systems such as BLAST®.

Cell Penetrating Conjugate with Functional Molecules

Identifying novel drugs to cure or prevent life threatening diseasesremains a highly active area of research. Most of such drugs tend tohave targets inside the cells and to reach the target they would have tocross the semi permeable membrane which is not straightforward orefficient in many cases. Therefore, developing novel mechanisms topenetrate the cell membrane remains desirable. On the other hand,scientific investigations and studies aiming to unravel various cellularmechanisms are also seeking to find novel methods of penetrating cellmembranes which can help in the labelling of the cell and its variousorganelles. Also, it would be highly useful in delivering the requiredmaterials inside cells for scientific experiments. Most of the cellpenetrating molecules described in recent reports involve theendocytosis mechanism of cell entry.

To circumvent the disadvantages of the intake of molecules throughendocytosis, for example the entrapment and degradation of drugs indifferent types of endosomal compartments which eventually fuse withdegradative compartment of cells such as lysosomes, a cell membranepenetrating conjugate is disclosed herein which can penetrate the cellmembrane to gain access to the inside the cells and can also be used todeliver various cargos including dyes, drugs, proteins, enzymes,antibodies, and nucleic acids inside the cells.

The present disclosure is not to be limited in scope by the specificembodiments described herein, which are intended for the purposes ofexemplification only. Functionally-equivalent products, compositions,and methods are clearly within the scope of the disclosure, as describedherein.

The present invention discloses a conjugate capable of penetrating acell membrane. The conjugate comprises a recombinant β helical protein,or a recombinant β helical protein portion, linked to a functionalmolecule wherein the protein has a longest dimension, defined as itslength, in the range of 5 nm-25 nm and a width or diameter, defined asthe dimension of the protein structure substantially perpendicular toits length, in the range of 1 nm-5 nm. The dimension of the protein indefined as measured in the solid state (characterized by x-raycrystallography or atomic force microscopy) or in solution state(dynamic light scattering measurements).

In embodiments of the present invention, the protein structure of theconjugate may have a longest dimension or length greater than 5 nm, 7.5nm, 10 nm, 11 nm, 12 nm, 13 nm or 14 nm. In embodiments, the proteinstructure of the conjugate may have a longest dimension or length lessthan 25 nm, 20 nm, 17.5 nm, 15 nm, 14 nm, 13 nm, 12 nm or 11 nm.Suitably the length is in the range of from 5 nm to 25 nm, more suitablyfrom 10 nm to 15 nm, even more suitably from 11 nm to 14 nm or 12 nm to13 nm. In embodiments, the protein structure of the conjugate may have awidth or diameter, in a dimension substantially perpendicular to itslength, of at least 1 nm, 1.1 nm, 1.2 nm, 1.3 nm, 1.4 nm, 1.5 nm, 1.6nm, 1.7 nm, 1.8 nm, 1.9 nm or 2.0 nm. In embodiments, the proteinstructure of the conjugate may have a longest dimension or length lessthan 5.0 mm, 4.5 nm, 4.0 nm, 3.5 nm, 3.0 nm, 2.9 nm, 2.8 nm, 2.7 nm, 2.6nm, 2.5 nm, 2.4 nm, 2.3 nm, 2.2 nm, 2.1 nm or 2.0 nm. Suitably the widthis in the range of from 1 nm to 5 nm, more suitably from 1 nm to 3 nm,even more suitably from 1.5 nm to 2.5 nm.

A surrogate for or an alternative definition for the physical size ofthe protein portion of the conjugate of the present invention is thecombination of its β-helical structure along with its molecular weight.The definition of the size of the protein based on its physicaldimensions or on its molecular weight may be used interchangeably. Inembodiments of the present invention, the molecular weight of theprotein portion may be at least 30 kDa. Suitably, the molecular weightof the protein portion may be at least 35 kDa, 40 kDa, 45 kDa or 50 kDa.In embodiments of the present invention, the molecular weight of theprotein portion may be at most 100 kDa. Suitably, the molecular weightof the protein portion may be at most 90 kDa, 80 kDa, 70 kDa, 60 kDa, 55kDa or 50 kDa. Suitably the molecular weight range of the proteinportion of the conjugate of the present invention is in the range of30-100 kDa, more suitably, 40-60 kDa, even more suitably 48-55 kDa.

Without wishing to be bound by theory, it is envisaged that thebeneficial properties in cell penetration demonstrated by the conjugatesof the present invention is due to the physical size of the proteinportion, as defined by the dimensions or molecular weight outlinedabove. A further feature of the proteins is the rigidity that deriveslargely from the uncommon β-helical secondary structure. Suitably theprotein may be more rigid than the membrane to be penetrated. Typicalcell membranes have a rigidity of 0.005 to 0.02 N/m² (as measured byAtomic Force Mircroscopy; Hayashi, “Tensile Properties and LocalStiffness of Cells”; Mechanics of Biological Tissue, pp 137-152)depending on cell type. Suitably the proteins of the present inventionhave a rigidity, or high stiffness parameter (K) typical of a β-helix of0.7 to 12 N/m² (Keten et al., Cell Mol. Bioeng. 2009; 2; 66-74, which isincorporated herein by reference).

A further feature thought to be influential in the efficiency of cellpenetration of the conjugates of the present invention is the chargeprofile and arrangement of the amino acids in the β-helical structure ofthe protein. Specifically, the presence of charged lysine, arginineasparagine, aspartic acid and/or glutamic acid ‘ladders’ in theβ-helical protein structure facilitates penetration of the cellmembrane, and the total negative charge of the protein sequence.

The term ‘ladder’ in respect of amino acid residues in β-helical proteinstructures is defined as a presence of alternatively arranged positively(lysine, arginine and/or asparagine) and negatively charged residues(aspartic acid and/or glutamic acid) along the length of the surface ofthe protein.

Without wishing to be bound by theory, the presence of charged ‘ladder’structures in the β-helical structure of the protein according to thepresent invention could be facilitating the interaction of the proteinwith the lipid molecules of the cell membrane (e.g. in forming hydrogenbond with the hydroxyl groups of the lipid molecules) and/or firstattachment of the protein onto the negatively charged cell membrane.

The total negative charge of the protein according to the presentinvention due to the negatively charged residues could be facilitatingthe repulsion leading to angular motion of the protein to make it standstraight on the membrane and puncture the membrane.

In embodiments of the present invention, the protein of the conjugatecomprises ladders of alternately arranged surface exposed positively andnegatively charged amino acid residues. In embodiments, the proteincomprises at least one of an arginine ladder (10-30 Arg residues), alysine ladder (10-30 Lys residues), an asparagine ladder (10-40 Asnresidues), aspartic acid (10-40 Asp residues) and glutamic acid (10-40Glu residues).

In embodiments of the present invention, the total formal charge of theprotein may be zero, or it may be non-zero. Suitably the total formalcharge is non-zero, more suitably the total formal charge is below zero(negative). In embodiments the total formal charge of the protein isbelow (i.e. more negative than) −10. Suitably, the total formal chargeof the protein is below −20, −25, −30, −35, −40, −45 or −50. Moresuitably the total form charge is below −20. In embodiments the totalformal charge of the protein is above (i.e. less negative than) −80.Suitably, the total formal charge of the protein is above −70, −65, −60,−55, −50, −45, −40, −35, or −30. More suitably, the total formal chargeis above −60. In embodiments, the total formal charge of the protein isin the range of from −10 to −80, more suitably from −20 to −60.

The efficiency of cell penetration and the mechanism of cell penetrationmay be modulated by optimizing the total formal charge of the conjugatesby increasing the number of positively charged residues (e.g. arginine)along the surface of the protein and the sequences can be mutated at theN terminal with the signalling sequences (SEQ ID NOs: 14, 15, 16 or 17and/or phosphatidyl choline) for attaining specificity of cellularorganelles or specificity of particular cancer cells.

In an embodiment, the protein of the conjugate of the present inventionsuitable for direct cell penetration (i.e. non-endocytotic) may have oneor more of the following structural parameters:

-   -   Beta helical structure with stiffness parameter K (beta helix)        0.2 to 12 N/m²;    -   Length between 5 nm to 25 nm;    -   Diameter between 1 nm to 5 nm;    -   Molecular weight between 25 KDa to 100 KDa;    -   Alternatively arranged ladders of surface exposed positive and        negative charge residues on the surface of the protein along the        length: an arginine ladder (10-30 Arg residues), a lysine ladder        (10-30 Lys residues), an asparagine ladder (10-40 Asn residues),        aspartic acid (10-40 Asp residues) and glutamic acid (10-40 Glu        residues);    -   Total formal charge (−20 to −60).

In embodiments of the present invention, the linker between theβ-helical protein and the functional molecule may be formed by a directlink between the β-helical protein and the functional molecule, forexample via an amide (or peptidic), or ester covalent linkage, or viametal coordination; or the linker may take the form of a linkermolecule. Suitably the link is by covalent or non-covalent bonds orinteractions. When the linker is a linker molecule the linker moleculemay take any suitable form that reversibly connects the β-helicalprotein and the functional molecule. In some embodiments, the linkermolecule may be selected from the group consisting of a peptide orprotein, PEG (polyethylene glycol) linker, organic molecule, metalconjugate, drug-metal conjugate, nucleic acid binding domains, andnucleic acid intercalating molecule.

In an embodiment, the present invention discloses a conjugate having arecombinant β helical protein of length in the range of 5 nm-25 nm,suitably, 10 nm-15 nm and width is in the range of 1 nm-5 nm, suitably 1nm-3 nm, wherein the β helical protein has a specific sequence linked toa functional molecule. Suitably the specific sequence is a penta-peptiderepeat. In an embodiment, the consensus sequence of the recombinant βhelical protein linked to a functional molecule is(STAV)₁(DN)₂(LF)₃(STR)₄(G)₅ (SEQ ID NO: 18). Examples of pent-peptiderepeat proteins are SEQ ID Nos: 1, 2, 6 and 8.

In embodiments of the present disclosure, the recombinant β helicalprotein is selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQID NO: 12.

In embodiments of the present invention, the functional molecule can beany organic molecule (anticancer drugs, antibiotics, NSAIDS, painrelieving drugs or any other drug molecule, fluorescent dyes,insecticides, pesticides etc.), drug-metal complex, metal, antibody,protein, polysaccharide, nucleic acids, peptides, nuclear localisingsignal, quantum dots and nanoparticles.

The conjugate can be used to transfer a functional molecule inside thecells facilitated by the ability of the conjugate to penetrate the cellmembrane. With appropriate localising signals associated with orintegrated with the conjugate or the functional molecule or both, theconjugate of the present invention can be used to target the functionalmolecule to a specific part of the interior of the cell, for example,the organelles present inside the cells.

In an embodiment, the present invention also discloses a process fortransferring a functional molecule inside the cells using the cellpenetrating conjugate of the present invention. The process disclosedcan further be used for cell labelling, cell penetration, and targetingof any functional molecule to cell organelles.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule, wherein the β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the β helical proteinis a pentapeptide-repeat protein.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the β helical proteincomprises tandemly repeated pentapeptide with consensus sequence

(SEQ ID NO: 18) (STAV)₁(DN)₂(LF)₃(STR)₄(G)₅

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule, wherein the β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein isrepresented by a sequence selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, and combinations thereof.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the β helical proteinis A1bG having a sequence as set forth in SEQ ID NO: 1.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the β helical proteinis EfsQNR having a sequence as set forth in SEQ ID NO: 2.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule, wherein the β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein is ananti-freeze protein having a sequence as set forth in SEQ ID NO: 3.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule, wherein the β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein is ananti-freeze protein having a sequence as set forth in SEQ ID NO: 4.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule, wherein the β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein is ananti-freeze protein having a sequence as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule, wherein the β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein isQNRB1 having a sequence as set forth in SEQ ID NO: 6.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule, wherein the β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein is UDPN-acetylglucosamine acyltransferase having a sequence as set forth inSEQ ID NO: 7.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule, wherein the β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein isNP275 having a sequence as set forth in SEQ ID NO: 8.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule, wherein the β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein ispectate lyase C having a sequence as set forth in SEQ ID NO: 9.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule, wherein the β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein is apectate lyase having a sequence as set forth in SEQ ID NO: 10.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule, wherein the β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein iscarbonic anhydrase having a sequence as set forth in SEQ ID NO: 11.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule, wherein the β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein isPectin Lyase A having a sequence as set forth in SEQ ID NO: 12.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the functionalmolecule is selected from the group consisting of dyes, drugs, metal,drug-metal complex, proteins, enzymes, antibodies, nucleic acids,polysaccharides, nuclear localizing signals, nanoparticles, andcombinations thereof.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the functionalmolecule is a dye.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the functionalmolecule is a drug.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the functionalmolecule is a metal.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the functionalmolecule is a drug-metal complex.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the functionalmolecule is a protein.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the functionalmolecule is an enzyme.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the functionalmolecule is an antibody.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the functionalmolecule is a nucleic acid.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the functionalmolecule is a polysaccharide.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the functionalmolecule is a nuclear localising signal.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the functionalmolecule is a nanoparticle.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the functionalmolecule is linked to the recombinant β helical protein by covalentbonds, non-covalent bonds, and combinations thereof.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the functionalmolecule is linked to the recombinant β helical protein by covalentbonds.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the functionalmolecule is linked to the recombinant β helical protein by non-covalentbonds.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule as described herein, wherein the complexfurther comprises a signal sequence.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule as described herein, wherein the conjugatefurther comprises a phospholipid molecule.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule as described herein, wherein the conjugatefurther comprises a signal sequence selected from the group consistingof SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule as described herein, wherein the conjugatefurther comprises a phosphatidyl choline molecule.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule as described herein, wherein the conjugatefurther comprises a signal sequence as set forth in SEQ ID NO: 14.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule as described herein, wherein the conjugatefurther comprises a signal sequence as set forth in SEQ ID NO: 15.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule as described herein, wherein the conjugatefurther comprises a signal sequence as set forth in SEQ ID NO: 16.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule as described herein, wherein the conjugatefurther comprises a signal sequence as set forth in SEQ ID NO: 17.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule for transferring the functional molecule tonucleus of the cell, wherein the β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the conjugate furthercomprises a signal sequence as set forth in SEQ ID NO: 14.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule for transferring the functional molecule tonucleus of the cell as described herein, wherein the β helical proteinis represented by a sequence selected from the group consisting of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, and combinations thereof, and wherein theconjugate further comprises a signal sequence as set forth in SEQ ID NO:14.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule for transferring the functional molecule tonucleus of the cell as described herein, wherein the functional moleculeis selected from the group consisting of dyes, drugs, metal, drug-metalcomplex, proteins, enzymes, antibodies, nucleic acids, polysaccharides,nuclear localizing signals, nanoparticles, and combinations thereof.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule for transferring the functional molecule toendoplasmic reticulum of the cell, wherein the β helical protein lengthis in the range of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in therange of 1 nm-5 nm, suitably 1 nm-3 nm, and wherein the conjugatefurther comprises a signal sequence as set forth in SEQ ID NO: 15.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule for transferring the functional molecule toendoplasmic reticulum of the cell as described herein, wherein the βhelical protein is represented by a sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and combinations thereof,and wherein the conjugate further comprises a signal sequence as setforth in SEQ ID NO: 15.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule for transferring the functional molecule toendoplasmic reticulum of the cell as described herein, wherein thefunctional molecule is selected from the group consisting of dyes,drugs, metal, drug-metal complex, proteins, enzymes, antibodies, nucleicacids, polysaccharides, nuclear localizing signals, nanoparticles, andcombinations thereof.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule for transferring the functional molecule tomitochondria of the cell, wherein the β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the conjugate furthercomprises a signal sequence as set forth in SEQ ID NO: 16.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule for transferring the functional molecule tomitochondria of the cell as described herein, wherein the β helicalprotein is represented by a sequence selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, and combinations thereof, and wherein theconjugate further comprises a signal sequence as set forth in SEQ ID NO:16.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule for transferring the functional molecule tomitochondria of the cell as described herein, wherein the functionalmolecule is selected from the group consisting of dyes, drugs, metal,drug-metal complex, proteins, enzymes, antibodies, nucleic acids,polysaccharides, nuclear localizing signals, nanoparticles, andcombinations thereof.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule for transferring the functional molecule toP-cadherin overexpressing breast cancer cells, wherein the β helicalprotein length is in the range of 5 nm-25 nm, suitably, 10 nm-15 nm andwidth is in the range of 1 nm-5 nm, suitably 1 nm-3 nm, and wherein theconjugate further comprises a signal sequence as set forth in SEQ ID NO:17.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule for transferring the functional molecule toP-cadherin overexpressing breast cancer cells as described herein,wherein the β helical protein is represented by a sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and combinationsthereof, and wherein the conjugate further comprises a signal sequenceas set forth in SEQ ID NO: 17.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule for transferring the functional molecule toP-cadherin overexpressing breast cancer cells as described herein,wherein the functional molecule is selected from the group consisting ofdyes, drugs, metal, drug-metal complex, proteins, enzymes, antibodies,nucleic acids, polysaccharides, nuclear localizing signals,nanoparticles, and combinations thereof.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule for transferring the functional molecule tomembrane of the cell, wherein the β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the conjugate furthercomprises a phosphatidyl choline molecule.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule for transferring the functional molecule tomembrane of the cell as described herein, wherein the β helical proteinis represented by a sequence selected from the group consisting of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, and combinations thereof, and wherein theconjugate further comprises a phosphatidyl choline molecule.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate comprising a recombinant β helical protein linkedto a functional molecule for transferring the functional molecule tomembrane of the cell as described herein, wherein the functionalmolecule is selected from the group consisting of dyes, drugs, metal,drug-metal complex, proteins, enzymes, antibodies, nucleic acids,polysaccharides, nuclear localizing signals, nanoparticles, andcombinations thereof.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell, said processcomprising: (a) linking the functional molecule to a recombinant βhelical protein to obtain a conjugate; (b) contacting the conjugate withat least one cell wherein contacting the conjugate transfers thefunctional molecule into the cell, and wherein length of the β helicalprotein is in the range of 5 nm-25 nm, suitably, 10 nm-15 nm and widthis in the range of 1 nm-5 nm, suitably 1 nm-3 nm. In an embodiment, theprocess further comprises after step (c), step (d) detecting thetransfer of the conjugate inside the cell.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is a pentapeptide-repeat protein.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein comprises tandemly repeated pentapeptidewith consensus sequence

(SEQ ID NO: 18) (STAV)₁(DN)(LF)₃(STR)₄(G)₅

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is represented by a sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and combinationsthereof.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is A1bG having a sequence as set forth inSEQ ID NO: 1.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is EfsQNR having a sequence as set forthin SEQ ID NO: 2.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is an anti-freeze protein having asequence as set forth in SEQ ID NO: 3.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is an anti-freeze protein having asequence as set forth in SEQ ID NO: 4.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is an anti-freeze protein having asequence as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is QNRB1 having a sequence as set forth inSEQ ID NO: 6.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is UDP N acetylglucosamine acyltransferasehaving a sequence as set forth in SEQ ID NO: 7.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is NP275 having a sequence is as set forthin SEQ ID NO: 8.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is pectate lyase C having a sequence asset forth in SEQ ID NO: 9.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is a pectate lyase having a sequence asset forth in SEQ ID NO: 10.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is carbonic anhydrase having a sequence asset forth in SEQ ID NO: 11.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein having a sequence is as set forth in SEQID NO: 12.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the functional molecule is selected from the group consisting ofdyes, drugs, metal, drug-metal complex, proteins, enzymes, antibodies,nucleic acids, polysaccharides, nuclear localizing signals,nanoparticles, and combinations thereof.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the functional molecule is a dye.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the functional molecule is a drug.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the functional molecule is a metal.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the functional molecule is a drug-metal complex.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the functional molecule is a protein.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the functional molecule is an enzyme.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the functional molecule is an antibody.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the functional molecule is a nucleic acid.

In an embodiment of the present disclosure, there is provided processfor transferring a functional molecule into a cell as described herein,wherein the functional molecule is a polysaccharide.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the functional molecule is a nuclear localising signal.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the functional molecule is a nanoparticle.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the functional molecule is linked to the recombinant β helicalprotein by covalent bonds, non-covalent bonds, and combinations thereof.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the cell is selected from the group consisting of eukaryoticcells, prokaryotic cells, and combinations thereof.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the cell is a prokaryotic cell.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the cell is a eukaryotic cell.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is represented by a sequence as set forthin SEQ ID NO: 1, and the functional molecule is NHS-coumarin dye.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is represented by a sequence as set forthin SEQ ID NO: 2, and the functional molecule is NHS-coumarin dye.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the β helical protein is represented by a sequence as set forthin SEQ ID NO: 2, and the functional molecule is ruthenium metal complex.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the process is used for cell-labelling.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule into a cell as described herein,wherein the process is used for delivering the functional molecules intothe cell.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule to an organelle of a cell, saidprocess comprising: (a) linking the functional molecule to a recombinantβ helical protein to obtain a conjugate; (b) further incorporating tothe conjugate any one signal sequence selected from the group consistingof SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; (c) contacting theconjugate of step (b) with at least one cell; wherein contacting theconjugate of step (b) transfers the functional molecule to the organelleinside the cell, wherein the organelle is selected from the groupconsisting of nucleus, endoplasmic reticulum, and mitochondria, andwherein the β helical protein length is in the range of 5 nm-25 nm,suitably, 10 nm-15 nm and width is in the range of 1 nm-5 nm, suitably 1nm-3 nm. In an embodiment, the process further comprises after step (c),step (d) detecting the transfer of the conjugate inside the cell.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule to an organelle of a cell asdescribed herein, wherein the recombinant β helical protein isrepresented by a sequence selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, and combinations thereof.

In an embodiment of the present disclosure, there is provided a processfor transferring the functional molecules to nucleus of the cell asdescribed herein, wherein the signal sequence is as set forth in SEQ IDNO: 14.

In an embodiment of the present disclosure, there is provided a processfor transferring the functional molecules to endoplasmic reticulum ofthe cell as described herein, wherein the signal sequence is as setforth in SEQ ID NO: 15.

In an embodiment of the present disclosure, there is provided a processfor transferring the functional molecules to mitochondria of the cell asdescribed herein, wherein the signal sequence is as set forth in SEQ IDNO: 16.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule to an organelle of a cell asdescribed herein, wherein the functional molecule is selected from thegroup consisting of dyes, drugs, metal, drug-metal complex, proteins,enzymes, antibodies, nucleic acids, polysaccharides, nuclear localizingsignals, nanoparticles, and combinations thereof.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule to P-cadherin-over expressingbreast cancer cells, said process comprising: (a) linking the functionalmolecule to a recombinant β helical protein to obtain a complex; (b)further incorporating a signal sequence as set forth in SEQ ID NO: 17 tothe complex; (c) contacting the complex of step (b) with at least oneP-cadherin-over expressing breast cancer cell; wherein contacting thecomplex of step (b) transfers the functional molecule into theP-cadherin-over expressing breast cancer cell, wherein the β helicalprotein length is in the range of 5 nm-25 nm, suitably, 10 nm-15 nm andwidth is in the range of 1 nm-5 nm, suitably 1 nm-3 nm. In anembodiment, the process further comprises after step (c), step (d)detecting the transfer of the complex inside the cell.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule to P-cadherin-over expressingbreast cancer cells as described herein, wherein the recombinant βhelical protein having a sequence is selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, and SEQ ID NO: 12.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule to P-cadherin-over expressingbreast cancer cells as described herein, wherein the functional moleculeis selected from the group consisting of dyes, drugs, metal, drug-metalcomplex, proteins, enzymes, antibodies, nucleic acids, polysaccharides,nuclear localizing signals, nanoparticles, and combinations thereof.

In an embodiment of the present disclosure, there is provided a processas described herein, wherein the process is used for targeted deliveryof the functional molecule into the cell.

In an embodiment of the present disclosure, there is provided a processas described herein, wherein the process is used for labelling of thecell.

In an embodiment of the present disclosure, there is provided a processas described herein, wherein the process is used for targeted deliveryof drugs in the cell.

In an embodiment of the present disclosure, there is provided a cellpenetrating complex as described herein, wherein the recombinant βhelical protein is used for delivering the functional molecule into thecell, and wherein the functional molecule is selected from the groupconsisting of dyes, drugs, metal, drug-metal conjugate, proteins,enzymes, antibodies, nucleic acids, polysaccharides, nuclear localizingsignals, nanoparticles, and combinations thereof.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule to actin protein present in acell, said process comprising: (a) linking the functional molecule to arecombinant β helical protein to obtain a conjugate; (b) furtherincorporating a signal sequence as set forth in SEQ ID NO: 22 to thecomplex; (c) contacting the conjugate of step (b) with at least onecell; wherein contacting the conjugate of step (b) transfers thefunctional molecule into the cell, wherein the β helical protein lengthis in the range of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in therange of 1 nm-5 nm, suitably 1 nm-3 nm. In an embodiment, the processfurther comprises after step (c), step (d) detecting the transfer of theconjugate inside the cell.

In an embodiment of the present disclosure, there is provided a processfor transferring a functional molecule to tubulin protein present in acell, said process comprising: (a) linking the functional molecule to arecombinant β helical protein to obtain a conjugate; (b) furtherincorporating a signal sequence as set forth in SEQ ID NO: 23 to theconjugate; (c) contacting the conjugate of step (b) with at least onecell; wherein contacting the conjugate of step (b) transfers thefunctional molecule into the P-cadherin-over expressing breast cancercell, wherein the β helical protein length is in the range of 5 nm-25nm, suitably, 10 nm-15 nm and width is in the range of 1 nm-5 nm,suitably 1 nm-3 nm. In an embodiment, the process further comprisesafter step (c), step (d) detecting the transfer of the conjugate insidethe cell.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the recombinant βhelical protein is used for cell penetration.

In an embodiment of the present disclosure, there is provided a cellpenetrating conjugate as described herein, wherein the recombinant βhelical protein is used for cell labelling.

Cell Penetrating Molecule with Nucleic Acid

In an embodiment, the present invention provides a solution to theproblem of delivery of nucleic acid fragments, as a functional molecule,that is faced by gene therapy treatments. The present document disclosesa conjugate comprising a recombinant β helical protein linked to anucleic acid molecule which can penetrate the cell membrane. In anembodiment, the recombinant β helical protein linked may be linked toone or more nucleic acid molecules, suitably a single nucleic acidmolecule, by a linker element, molecule, portion or moiety.

The conjugate comprising recombinant β helical protein, a linker and aplasmid is shown to successfully penetrate the cell membrane toestablish the expression of a gene forming the part of the plasmid. Theconjugate avoids the pathway of endocytosis and crosses the cellmembrane by directly penetrating the membrane. The conjugate henceovercomes the problems faced by entry through endocytosis and at thesame time effectively penetrating the cell membrane.

In an embodiment, the present invention discloses a conjugate capable ofpenetrating cell membrane. In some embodiments, the conjugate comprisesa recombinant β helical protein of length in the range of 5 nm-25 nm,suitably, 10 nm-15 nm and width is in the range of 1 nm-5 nm, suitably 1nm-3 nm linked to a nucleic acid molecule via a linker. In anembodiment, the recombinant β helical protein can be a pentapeptiderepeat protein have a consensus sequence (STAV)₁(DN)₂(LF)₃(STR)₄(G)₅(SEQ ID NO: 18). In another embodiment of the present disclosure, therecombinant β helical protein is selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, and SEQ ID NO: 12.

According to the present disclosure, in an embodiment the recombinant βhelical protein is linked to a nucleic acid molecule by a linkerelement, molecule, portion or moiety. In embodiments, the linker elementmay be a direct link by covalent or non-covalent bonds. When the linkeris a linker molecule it may be selected from the group consisting ofprotein, metal conjugate, drug-metal conjugate, DNA binding domain, andnucleic acid intercalating molecule. In an embodiment, the nucleic acidmolecule comprising the part of the conjugate comprises at least onegene of interest. In an embodiment, the nucleic acid molecule is linkedto a complex comprising the recombinant β helical protein and the linkerto form the conjugate as disclosed in the present invention. In anembodiment, the conjugate is able to enter the cell by penetrating thecell membrane and a gene forming a part of the nucleic acid molecule isable to express inside the cells.

The present invention also discloses a process for transferring anucleic acid molecule inside the cells using the conjugate of thepresent invention. In an embodiment, the process disclosed can furtherbe used for gene therapy techniques for facilitating non-viralapproaches for delivering the gene of interest in a cell, therebyeffectively compensating for a defective gene or protein inside thecell.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein isrepresented by a sequence selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, and combinations thereof.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein is apentapeptide repeat protein.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical proteincomprises tandemly repeated pentapeptide with consensus sequence

(SEQ ID NO: ₁₈) (STAV)₁(DN)(LF)₃(STR)₄(G)₅.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the linker is a linkermolecule selected from the group consisting of protein, metal conjugate,drug-metal conjugate, DNA binding domain, nucleic acid intercalatingmolecule, and combinations thereof.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein isrepresented by a sequence selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, and combinations thereof, and wherein the linkeris a linker molecule selected from the group consisting of protein,metal conjugate, drug-metal conjugate, DNA binding domain, nucleic acidintercalating molecule, and combinations thereof.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the linker is linked to therecombinant β helical protein by covalent bonds, non-covalent bonds, andcombinations thereof.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the linker is a linkermolecule selected from the group consisting of protein, metal conjugate,drug-metal conjugate, DNA binding domain, nucleic acid intercalatingmolecule and combinations thereof, and wherein the linker is linked tothe recombinant β helical protein by covalent bonds, non-covalent bonds,and combinations thereof.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the nucleic acid moleculecomprises at least one gene of interest.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein isrepresented by a sequence selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, and combinations thereof, and wherein the nucleicacid molecule comprises at least one gene of interest.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein isrepresented by a sequence selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, and combinations thereof, and wherein the linkeris a linker molecule selected from the group consisting of protein,metal conjugate, drug-metal conjugate, DNA binding domain, nucleic acidintercalating molecule and combinations thereof, and wherein the nucleicacid molecule comprises at least one gene of interest.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein is A1bGhaving a sequence as set forth in SEQ ID NO: 1.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein isEfsQNR having a sequence as set forth in SEQ ID NO: 2.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein is ananti-freeze protein having a sequence as set forth in SEQ ID NO: 3.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein is ananti-freeze protein having a sequence as set forth in SEQ ID NO: 4.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein is ananti-freeze protein having a sequence as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein isQNRB1 having a sequence as set forth in SEQ ID NO: 6.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein is UDPN acetylglucosamine acyltransferase having a sequence as set forth inSEQ ID NO: 7.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein isNP275 having a sequence as set forth in SEQ ID NO: 8.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein ispectate lyase C having a sequence as set forth in SEQ ID NO: 9.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein is apectate lyase having a sequence as set forth in SEQ ID NO: 10.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein iscarbonic anhydrase having a sequence as set forth in SEQ ID NO: 11.

In an embodiment of the present disclosure, there is provided aconjugate comprising: (a) at least one recombinant β helical protein;(b) at least one linker; and (c) at least one nucleic acid molecule,wherein the at least one recombinant β helical protein length is in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β helical protein isPectin Lyase A having a sequence as set forth in SEQ ID NO: 12.

In an embodiment of the present disclosure, there is provided a processfor transferring a nucleic acid molecule into a cell, said processcomprising: (i) linking the nucleic acid molecule to a complexcomprising: (a) at least one recombinant β helical protein; and (b) atleast one linker to obtain a conjugate; and (ii) contacting theconjugate to at least one cell, wherein contacting the conjugatetransfers the nucleic acid molecule into the cell, and wherein therecombinant β helical protein length is in the range of 5 nm-25 nm,suitably, 10 nm-15 nm and width is in the range of 1 nm-5 nm, suitably 1nm-3 nm.

In an embodiment of the present disclosure, there is provided a processfor transferring a nucleic acid molecule into a cell as describedherein, wherein the β helical protein is represented by a sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, andcombinations thereof.

In an embodiment of the present disclosure, there is provided a processfor transferring a nucleic acid molecule into a cell as describedherein, wherein the β helical protein is a pentapeptide repeat protein.

In an embodiment of the present disclosure, there is provided a processfor transferring a nucleic acid molecule into a cell as describedherein, wherein the β helical protein comprises tandemly repeatedpentapeptide with consensus sequence

(SEQ ID NO: 18) (STAV)₁(DN)₂(LF)₃(STR)₄(G)₅.

In an embodiment of the present disclosure, there is provided a processfor transferring a nucleic acid molecule into a cell as describedherein, wherein the linker is a linker molecule selected from the groupconsisting of protein, metal conjugate, drug-metal conjugate, DNAbinding domain, nucleic acid intercalating molecule, and combinationsthereof.

In an embodiment of the present disclosure, there is provided a processfor transferring a nucleic acid molecule into a cell as describedherein, wherein the β helical protein is represented by a sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, andcombinations thereof, and wherein the linker is a linker moleculeselected from the group consisting of protein, metal conjugate,drug-metal conjugate, DNA binding domain, nucleic acid intercalatingmolecule, and combinations thereof.

In an embodiment of the present disclosure, there is provided a processfor transferring a nucleic acid molecule into a cell as describedherein, wherein the linker is linked to the recombinant β helicalprotein by covalent bonds, non-covalent bonds, and combinations thereof.

In an embodiment of the present disclosure, there is provided a processfor transferring a nucleic acid molecule into a cell as describedherein, wherein the linker is a linker molecule selected from the groupconsisting of protein, metal conjugate, drug-metal conjugate, DNAbinding domain, nucleic acid intercalating molecule and combinationsthereof, and wherein the linker is linked to the recombinant β helicalprotein by covalent bonds, non-covalent bonds, and combinations thereof.

In an embodiment of the present disclosure, there is provided a processfor transferring a nucleic acid molecule into a cell as describedherein, wherein the cell is either a prokaryotic cell or a eukaryoticcell.

In an embodiment of the present disclosure, there is provided aconjugate as described herein, wherein the conjugate is used as atransfecting agent.

In an embodiment of the present disclosure, there is provided aconjugate as described herein, wherein the conjugate is used for genetherapy.

Examples—Cell Penetrating Molecule with Functional Molecules

The disclosure will now be illustrated with working examples, which isintended to illustrate the working of disclosure and not intended totake restrictively to imply any limitations on the scope of the presentdisclosure. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood to one ofordinary skill in the art to which this disclosure belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice of the disclosed processes and compositions,the exemplary processes, devices and materials are described herein. Itis to be understood that this disclosure is not limited to particularmethods, and experimental conditions described, as such processes andconditions may vary.

In the following examples, methods and protocols for carrying outplasmid and protein studies have been provided. Also provided are theprotocols for obtaining the conjugate, labelling of the protein, andmethods of carrying out cell penetration studies using protein-drugconjugate and protein-label conjugate. The results section specificallydescribes the proof-of-concept of the cell penetration ability of theconjugate as disclosed in the present invention. The in-vitro labellingof different cell lines has been carried out using the conjugate of thepresent invention as per the method disclosed. The assay with adrug-protein conjugate have also been carried out to study the abilityof the conjugate in enhancing the drug uptake by cell penetration.

Material and Methods

The dye Hoechst 33342 was procured from Invitrogen; NHS-coumarin, andATTO 520-NHS, ATTO 390-NHS and ATTO 647N-NHS were procured from Sigma.The organic solvents and reagents used for UV-visual spectrophotometryand CD spectra were procured from Sigma and Merck. The reagents forstudying the expression of plasmids and purification of proteins wereprocured from Sigma Aldrich and Merck. The reagents required for MTTassay were procured from MP Biomedicals. Ruthenium metal complex wasprocured from Sigma.

Example 1

Plasmid/Protein studies—The plasmid containing A1bG gene as shown in SEQID NO: 19, was obtained by a method as published previously (Vetting etal. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2011: 67(3):296-302, the contents of which is incorporated herein by reference andspecific details provided below).

The open reading frame of A1bG was amplified by standard PCR techniquesusing X albilineans (ATCC 29184; Pieretti et al., BMC Genomics, 2009,10:616, 1-15) chromosomal DNA as a template. The oligonucleotides A1bGF(5′-ATCCCGCTCATATGCCGGCCAAGACCCTTG-3′; SEQ ID NO: 26) and A1bGR(5′-ATCCCGCTCTCGAGTCAATCGGACAGCTCGATATC-3′; SEQ ID NO: 27) containingNdeI and XhoI restriction sites, respectively, were used. The PCRfragment was cloned into pET-28a(+) and recombinant A1bG bearing athrombin-cleavable N-terminal His6 tag was expressed in E. coli strainBL21 (DE3). For shake-flask growth, 1 liter of Luria broth mediumsupplemented with kanamycin (35 μg/ml) was inoculated with 10 ml of anovernight culture and incubated at 37° C. The culture was grown tomid-log phase (A₆₀₀ of ˜0.8), cooled to 20° C., induced with 0.5 mM IPTGand further incubated overnight at 20° C. All purification procedureswere carried out at 4° C. The cells were collected by centrifugation at3000 g, re-suspended in buffer A [50 mM Tris-HCl pH 7.8 containing 300mM NaCl, protease inhibitors, lysozyme (5 μg/ml) and DNase I (0.1μg/ml)] and stirred for 20 min. The cells were then lysed by sonicationand cell debris was removed by centrifugation at 10 000 g for 30 min.The supernatant was loaded onto a nickel-nitrilotriacetic acid (Ni-NTA)column pre-equilibrated with buffer A and washed with ten column volumesof the same buffer. The bound proteins were eluted with a linear β to0.3 M imidazole gradient and the peak fractions were pooled andconcentrated.

The plasmid containing EfsQNR gene as shown in SEQ ID NO: 20 wasobtained by a method as published previously (Hegde et al., Antimicrob.Agents Chemother. 2011 January; 55(1): 110-117, the contents of which isincorporated herein by reference and details provided below).

The open reading frame of EfsQNR was amplified by standard PCRtechniques using E. faecalis V583 (ATCC 700802; ENTFA 226185)chromosomal DNA as the template. The oligonucleotides(5′-ATCCCGCTCATATGAAAATAACTTATCCCTTGCCA-3; SEQ ID NO: 28) and(5′-ATCCCGCTCTCGAGTTAGGTAATCACCAAACCAAGT-3; SEQ ID NO: 29), containingNdeI and XhoI restriction sites, respectively, were used. The PCRfragment was cloned into pET-28a(+), and the recombinant EfsQNR bearinga thrombin-cleavable N-terminal His6 tag was expressed in E. coli strainBL21(DE3). For shake flask growth, 1 liter of Luria broth mediumsupplemented with kanamycin (35 μg/ml) was inoculated with 10 ml of anovernight culture and incubated at 37° C. The culture was grown tomid-log phase (A₆₀₀˜0.8), cooled to 20° C., induced with 0.5 mMisopropyl-β-d-thiogalactopyranoside (IPTG), and further incubatedovernight at 20° C.

The cells were collected by centrifugation at 1,200 g and re-suspendedin buffer A (50 mM Tris-HCl [pH 7.8], 300 mM NaCl) containing proteaseinhibitors, lysozyme (5 μg/ml), and DNase I (0.1 μg/ml), and the mixturewas stirred for 20 min. The cells then were lysed by sonication, andcell debris was removed by centrifugation at 10,000 g for 30 min. Thesupernatant was loaded onto a Ni-NTA column pre-equilibrated with bufferA and washed with 10 column volumes of the same buffer. The boundproteins were eluted with a linear β to 0.3 M imidazole gradient withfractions pooled and concentrated.

As a comparative example, a plasmid containing TtCuA gene as shown inSEQ ID NO: 13 was obtained by a method in Biochemistry, 2008, 47,1309-1318, which is incorporated herein by reference.

Example 2

Toxicity assay—The industry-standard MTT assay (ex-Sigma Aldrich) wasperformed to analyse the toxicity of β-helical protein-cytotoxic drugconjugates against mammalian cells, for example HeLa cells. The cellswere cultured by using standard protocol. One million cells were seededin confocal plates (1 cm dish), grown for 6-8 h. A mixture of thecytotoxic drug and the EfsQNR protein (mixed at a ratio of 2:1) wasadded to the cells and incubated at room temperature for 15 minutes to24 h to 72 h. After the incubation, the cells were washed with PBS andtreated with MTT and further incubated for 24 hours. The cells were thenwashed and analysed for absorbance values at 570 nm. Viable cells withactive metabolism convert MTT into a purple coloured formazan productwith an absorbance maximum near 570 nm. Dead cells cannot convert MTTinto formazan therefore, by analysing the absorbance values at 570 nmpercentage of viable cells can be calculated for a given protein or anyother molecule.

Example 3

Labelling of proteins with fluorescent dyes—Fluorescent dyes wereconsidered as an example of a functional molecule for investigating thecell membrane penetration ability of the conjugate. Labelling of therecombinant protein with a dye was carried out by performing a series ofreaction in dark conditions. The protein to be labelled was collected inPBS buffer (1X and pH 7.3) and the dye was collected at two to threetimes higher concentration to that of the protein. The protein was addedto 0.1M sodium carbonate buffer (pH 8.5) followed by the dye. The dyewas added very slowly (3 μl each time) to the buffer containing theprotein kept on ice accompanied by occasional shaking. The resultingsolution was wrapped with aluminium foil to keep away any light. Thesolution was stirred for 1 hour at room temperature and was thenpurified by gel filtration, i.e. passed through a desalting column or aPD 10 column with 1×PBS buffer. The resulting labelled protein wascharacterized using UV-visual spectrophotometry to determine dye:protein ratio.

Example 4

Cellular uptake of labelled proteins—The uptake of labelled proteins waschecked by treating mammalian cells with different labelled proteinssuch as A1bG (SEQ ID NO: 1), EfsQNR (SEQ ID NO: 2) and, as a comparativeexample, TtCuA (SEQ ID NO: 13). TtCuA is a cytochrome oxidase c proteinfrom the organism Thermus thermophilus. It is represented by the aminoacid sequence as set forth in SEQ ID NO: 13. The cells were seeded at aconcentration of one million in confocal plates (1 cm dish) and allowedto grow for 6-8 hours. Subsequently, the labelled proteins were added tothe cells at different concentrations and incubated under standardconditions of 5% CO₂ and 37° C. The cells were then washed twice, after3 hours and 24 hours with HBSS (Hanks blank salt solution) or PBS andobserved under fluorescence microscopy and/or confocal laser scanningmicroscope.

The uptake of labelled proteins was checked by treating E-Coli and yeast(Kluveromyces) cells with different labelled proteins such as A1bG (SEQID NO: 1), EfsQNR (SEQ ID NO: 2). E. coli or Kluveromyces cells wereinoculated and grown overnight until OD reached 0.6. The cells werediluted to 50 times and 3 micromolar labelled protein (conjugate) wasadded. The cells were further grown under standard growth condition (37°C. shaking condition) for 24 h. The treated cells were then centrifugedand washed in PBS for 3-4 times. Then the cells were dispersed in 100microliter PBS and then imaged in fluorescence/confocal microscope.

Example 5

Cellular uptake of drug—HeLa cells were used for determining theenhanced uptake of the drug using the cell penetrating conjugate of thepresent invention. HeLa cells were seeded at a concentration of 10,000in a 96-well plate and allowed to grow for 6-8 hours. Ruthenium metalcomplex and EfsQNR were mixed in different ratios of 1:1, 1:2, and 1:3to form a conjugate, which was subsequently added to the cells. Theconjugate formed of ruthenium to EfsQNR in a ratio of 1:2 yielded anenhanced result. The cell death after 24 hours was monitored by MTTassay as described previously and was correlated to the percentageuptake of ruthenium-EfsQNR conjugate by HeLa cells.

Results of Examples 1 to 5 Characterization of Proteins and Conjugates

Structural specificity—The size of the A1bG protein was determined fromthe published crystal structure pdb id: 2xt2.pdb. By measuring the endto end atom distance of dimeric structure in Pymol®, the length wasdetermined to be 10.7 nm, and the width (diameter) to be 2.6 nm. Thetotal formal charge was −27. There are alternatively arranged positiveand negative charge residues: Presence of an arginine ladder (total of20 Arg residues), Lysine (14 residues) and aspartic acid (30 Aspresidues) and glutamic acid (31 Glu residues). Asparagine (20 Asnresidues) ladder along the surface of the protein.

The size of the EfsQNR protein was determined from the published crystalstructure pdb id: 2w7z.pdb. By measuring the end to end atom distance ofthe dimeric structure in Pymol®, the length of the protein wasdetermined to be 10.9 nm, and the width (diameter) to be 2.8 nm. Totalformal charge −41. Alternatively arranged positive and negative chargeresidues: Presence of an arginine ladder (total of 16 Arg residues),Lysine (12 residues) and aspartic acid (25 Asp residues) and glutamicacid (33 Glu residues). Asparagine (33 Asn residues) ladder along thesurface of the protein.

For comparison, the size of the TtCuA protein was determined from thepublished crystal structure pdb id: 2CuA.pdb. By measuring the end toend atom distance of structure in Pymol®, the length was determined tobe 3.3 nm, and the width (diameter) to be 1.9 nm. The structurecomprises 5 Arginine residues, 4 Lysine residues, 9 Glutamic acidresidues and 6 Asparagine residues with a total charge of −3.

FIG. 1 displays the CD spectra of purified A1bG protein (SEQ ID NO: 1)used for the study. Protein was dialyzed in PBS and 1-5 micromolarconcentration was used to measure the CD spectroscopy in 200-300 nmregion. Further dilution was done with PBS wherever needed. The negativevalue of CD at 220 nm for this protein indicates arrangement of β sheets(β barrel or β helical structures).

FIG. 2 shows the purified plasmids used for expressing β helicalproteins, A1bG and EfsQNR, each showing identical results in triplicate(A1bG-1 to A1bG-3 and EfsQNR-1 to EfsQNR-3).

FIG. 3 shows the purified protein bands of the A1bG and EfsQNR proteinsin a polyacrylamide gel. After performing SDS-gel electrophoresis, itwas noted that molecular weight of both the proteins in monomer formswere approximately 30 kDa. The approximate molecular weight in dimerforms of A1bG and EfsQNR is around 48 kDa. The proteins, A1bG and EfsQNRwere used to form a conjugate with a functional molecule, which was thenused to study the cell penetrating efficacy. The proteins were furthertested for any cytotoxicity using HeLa cells for determining the courseof study (see “Cytotoxicity Studies” below).

FIG. 4 shows the result of a MALDI TOF mass spectrometry analysis of theEfsQNR protein and the A1bG protein. For this experiment, the proteinwas dialyzed in water and sample was prepared using sinapic acid asmatrix with 0.1% TFA and acetonitrile. 1-10 micromolar of protein wasused. The results show the EfsQNR protein having a molecular weight ofapproximately 26.5 kDa, and the A1bG protein having a molecular weightof approximately 23 kDa.

Cytotoxicity Studies

MTT assay as described previously was employed to study toxicity of A1bGand EfsQNR proteins against HeLa cells. For the toxicity assay, samplesof Placebo, control, A1bG (30 μM) and EfsQNR (30 μM) were used intriplicates. Table 1 depicts the absorbance values at 570 nm of eachsample after treatment with MTT. On observing the values, it can beappreciated that the cells treated with 30 μM of A1bG and 30 μM ofEfsQNR display 95% viability as compared to the control cells. Thisclearly suggests that the tested protein at the tested concentration isnot toxic for HeLa cells and hence is a safe concentration to use infurther experiments.

Table 1 presented here depicts the absorbance values at 570 nm in MTTassay for evaluating toxicity of the proteins:

TABLE 1 1 2 3 4 5 6 7 8 9 10 11 12 PLACEBO CONTROL EfsQNR AlbG 0.0490.05 0.046 1.341 1.348 1.413 0.901 1.111 0.971 1.002 1.03 0.932

The placebo for this experiment was 20 mM Tris buffer of pH 8.0,whereas, the control considered was only HeLa cells without adding anyprotein or other substrate.

Labelling Studies

FIG. 4 shows the UV-visual spectrophotometry of the labelled A1bG andEfsQNR conjugates by the procedure as mentioned previously (see“Labelling of proteins with fluorescent dyes” above). The UV-visualspectra was analysed to calculate the labelling ratio of bothconjugates. The labelling ratio signifies the number of dyes attachedper protein molecule. This is calculated by measuring the ratio betweenthe absorbance values of the dye (at a wavelength depending on the dyeused for labelling) and the absorbance of the protein at 280 nm. The dyeused for labelling in this experiment was NHS-coumarin. In case of A1bGprotein, the dye: protein molecule ratio is observed to be 1.95. In caseof EfsQNR the dye: protein molecule ratio is found to be 5.5. The UV-visspectra confirmed the labelling of both A1bG and EfsQNR proteins withNHS-coumarin dye. The labelled proteins were further used to establishcell penetration in this study.

Cellular Penetration Studies

FIG. 5 shows the fluorescence microscopic images of HeLa cells treatedwith NHS-C labelled A1bG, EfsQNR, and TtCuA protein (conjugates). Theintake of the labelled proteins (conjugates) was compared with theuntreated HeLa cells which were considered as a control set for thisstudy. Hoechst Blue is a nuclear labelling dye used along with NHS-C dyewhich is used for labelling the proteins. The proteins A1bG and EfsQNRare β helical proteins of length in the range of 5 nm-25 nm, suitably,10 nm-15 nm and width is in the range of 1 nm-5 nm, suitably 1 nm-3 nmrepresenting the recombinant β helical protein of the present invention.However, TtCuA is a protein of length around 4 nm and is composed of βstrands forming β barrels, thereby representing a comparative examplefor the recombinant β helical protein of the present invention. Thesetwo different categories of proteins were considered for this study toestablish the superior cell penetrating ability of the conjugate of thepresent invention as compared to different type of β helical protein.

In FIG. 5 , panel A represents the control cells which are treated onlywith nuclear labelling dye (Hoechst Blue) signifying the lack of lighterdye, panel B represents the cells treated with Hoechst Blue dye and withTtCuA-NHSC labelled protein (conjugate), panel C represents the cellstreated with Hoechst Blue dye and A1bG-NHSC labelled protein (conjugate)and panel D represents the cells treated with Hoechst Blue dye andEfsQNR-NHSC labelled protein (conjugate). On observing the figure, itcan be appreciated that significantly less fluorescence can be seeninside the cells as shown in panel B signifying less presence ofTtCuA-NHSC conjugate inside these cells, whereas greater fluorescencecan be seen inside the cells represented in panel C and panel Dsignifying larger presence of A1bG-NHSC conjugate and EfsQNR-NHSCconjugate inside the cells. Therefore, it can be appreciated thatTtCuA-NHSC conjugate is not able to effectively penetrate the cellmembrane to gain entry inside cells as compared to A1bG-NHSC conjugateand EfsQNR-NHSC conjugate.

On comparing panels C and D, it can be observed that EfsQNR-NHSCconjugate is able to penetrate the membrane more efficiently as comparedto the A1bG-NHSC conjugate. Therefore, the cell penetration ability ofthe three conjugates can be summarised in an increasing order of celluptake as TtCuA-NHSC <A1bG-NHSC <EfsQNR-NHSC. This further proves thecell penetrating ability of the conjugate comprising a recombinant βhelical protein of length and breadth in the range of 5 nm-25 nm,suitably, 10 nm-15 nm and width is in the range of 1 nm-5 nm, suitably 1nm-3 nm respectively. The conjugates independently comprising A1bG andEfsQNR are able to show enhanced cell permeability as compared to theconjugate comprising TtCuA.

Since the conjugate comprising EfsQNR protein displayed the highestability to penetrate cell membrane, EfsQNR protein was further studiedwith different dyes for enhanced labelling of mammalian cells.

FIG. 22 shows in real-time cell entry proceeding via direct interactionof the conjugate with the cell-membrane. To visualise the cells underfluorescence microscopy they were treated with the dye Hoechst 33342(blue, appearing dark in in FIG. 22 ) which binds to the nucleus and thedye Alexa 594-WGA (red, appearing lighter in in FIG. 22 ) which binds tothe plasma membrane indicating that the EfsQNR-ATTO-520NHS (green,appearing as light circles in FIG. 22 ) efficiently penetrates insidethe cell by a direct mechanism avoiding any endocytotic mechanism.

Labelling of Mammalian Cells

FIG. 7 depicts the ability of a conjugate comprising EfsQNR conjugatedwith a dye in labelling of HeLa cells. FIG. 6 depicts the cells labelledwith EfsQNR-ATTO-520NHS prepared according to the method of labellingabove. The dye Hoechst 33342 (blue, appearing dark in in FIG. 7 ) bindsto the nucleus and the dye Alexa 594-WGA (red, appearing lighter in inFIG. 7 ) binds to the plasma membrane. The conjugate is formed bylinking EfsQNR protein with a green fluorescent dye ATTO-520NHS(appearing as the lightest dye in FIG. 7 ). It can be clearly observedthat the EfsQNR-ATTO-520NHS conjugate penetrates the HeLa cells to leadto efficient labelling (Panel B) of the cells when compared to thecontrol panel A showing HeLa cells which are not treated with theconjugate.

FIG. 8 depicts a similar result for HeLa cells treated with theconjugate formed by linking EfsQNR protein with a blue fluorescent dyeATTO-390NHS (appearing lighter in FIG. 8 )

FIG. 9 depicts a similar result for microglial cells labelled with theEfsQNR-ATTO-520NHS which was prepared according to the method oflabelling above. Panel A shows significant fluorescence of the dye frominside the cells. Panel B shows microglial cells additionally treatedwith the dye Hoechst 33342 (blue, appearing dark in in FIG. 9 ) whichbinds to the nucleus and the dye Alexa 594-WGA (red, appearing lighterin in FIG. 9 ) which binds to the plasma membrane indicating that theEfsQNR-ATTO-520NHS has efficiently penetrated inside the cell.

FIG. 10 depicts a similar result for keratinocyte cells labelled withthe conjugate EfsQNR-ATTO-520NHS which was prepared according to themethod of labelling above. Panel A shows significant fluorescence of thedye from inside the cells. Panel B shows keratinocyte cells additionallytreated with the dye Hoechst 33342 (blue, appearing dark in in FIG. 10 )which binds to the nucleus indicating that the EfsQNR-ATTO-520NHS hasefficiently penetrated inside the cell to be present near the nucleus.

FIG. 11 depicts a similar result for SH-SYSY cells labelled with theconjugate EfsQNR-ATTO-520NHS which was prepared according to the methodof labelling above.

FIG. 12 depicts a similar result for Mouse ES cells labelled with theEfsQNR-ATTO-520NHS which was prepared according to the method oflabelling above. FIG. 12 shows Mouse ES cells treated with the dyeHoechst 33342 (blue, appearing dark in in FIG. 12 ) which binds to thenucleus and the dye Alexa 594-WGA (red, appearing lighter in in FIG. 12) which binds to the plasma membrane indicating that theEfsQNR-ATTO-520NHS has efficiently penetrated inside the cell.

Labelling of Non-Mammalian Cells

FIGS. 13 and 14 depict the ability of a conjugate comprising EfsQNRconjugated with a dye in labelling of E. coli and yeast (kluveromyces)cells. FIGS. 13 and 14 depict respectively E. coli and yeast(kluveromyces) cells labelled with EfsQNR-ATTO-520NHS prepared accordingto the method of labelling above.

FACS Sorting of HeLa Cells Treated with the Conjugate

FIG. 15 shows the results of standard FACS sorting of HeLa cells treatedwith a conjugate labelled with the conjugate EfsQNR-ATTO-647N. Treatmentof cells for only 10 minutes (Panel B) leads to significant uptake ofthe dye compared to the control (Panel A) which continues to increase at1 h (Panel C) up to the measured 3 h maximum (Panel D).

Drug Uptake Studies

FIG. 16 depicts the cellular uptake of the ruthenium metal complexRu(CO)₃Cl glycinate in HeLa cells. The ruthenium metal complex wasconjugated with EfsQNR protein to form a conjugate consisting ofruthenium metal complex-EfsQNR. The HeLa cells were treated with theruthenium metal complex-EfsQNR conjugate as described above (see“Cellular penetration studies” above) and uptake of the complex wasdetected by checking the viability of the cells by performing MTT assayas described previously. On observing the graph (FIG. 14 ), it can beappreciated that upon treatment with only ruthenium metal complex, thecellular uptake is only 0.5%, whereas upon treatment with rutheniummetal complex-EfsQNR protein conjugate the cellular uptake of thecomplex increases to 24.4%. Therefore, it can be ascertained that theconjugate comprising EfsQNR protein facilitated 50-fold higher cellularuptake as compared to ruthenium metal complex alone. This enhanceduptake can be further exploited by using the conjugate of the presentinvention to deliver many life-saving drugs to the target therebyreducing the dose of the drug and subsequently decreasing theside-effects.

FIG. 17 shows the results of a viability study of mammalian cells (HeLaand HepG2) cultured with a conjugate comprising EfsQNR protein linked tothe chemotherapy drug, Cisplatin® (cisplatinum orcis-diamminedichloroplatinum(II) (CDDP)) labelled ‘CPDD’ in FIG. 17 .Mammalian cells (HeLa: Panel A; or HepG2: Panel B) were cultured byusing standard protocol. One million cells were seeded in confocalplates (1 cm dish), grown for 8 h. A conjugate comprising Cisplatin® andEfsQNR (prepared in accordance with the method described above in amolar ratio of 2:1) was then added and the culture kept at roomtemperature for 15 minutes. The cells were kept under standard growthcondition for 24 h and 72 h. Then the cells were washed with PBS and MTTsolution was added. After the formazan dyes are formed it was dissolvedin DMSO and absorbance at 550 nm was measured. It is apparent from theresults that in both cell types tested similar cytotoxic efficacy ofCisplatin® is demonstrated with a lower dose when the drug is deliveredas a conjugate in accordance with the present invention.

It is noted that in all experiments where tested, similar results wereobtained when the EfsQNR protein was replaced with the A1bG protein.

Advantages

Overall, it can be concluded that a conjugate comprising a recombinant βhelical protein of specific length and width, and a functional moleculecan act as a highly efficient cell membrane penetrating conjugate. Theability of the conjugate to directly penetrate the cell membraneprovides advantages as compared to the endocytotic entry mechanism.Working examples of the A1bG-NHSC and EfsQNR-NHSC conjugates areprovided in the present disclosure which demonstrate their ability topenetrate the cell membrane. The EfsQNR-dye conjugate is shown toenhance the ability of labelling of a variety of mammalian andnon-mammalian cells because of its ability to effectively penetrate thecell membrane. The conjugate comprising EfsQNR and/or A1bG has beenshown to increase the cellular uptake of drugs including ruthenium metalcomplexes and Cisplatin® in HeLa and HepG2 cells. This ability can befurther exploited to elevate the uptake of different anti-cancer drugsby cancer cells which can facilitate the treatment by selectivelytargeting the cancerous cells in an efficient manner and simultaneouslywith a reduced requirement of the drug. Reduction in the dose ofanti-cancer drug can also circumvent the side-effects associated withadministration of such drugs.

Examples—Cell Penetrating Molecule with Nucleic Acid

In the following paragraphs, working examples have been provided fordelivering a nucleic acid having a gene of interest inside the cell bypenetrating the cell membrane. The delivery is made possible because ofthe use of a conjugate as an embodiment of the present invention whichfurther comprises a nucleic acid molecule. The examples also depict theexpression of the gene of interest after penetrating the cell membraneto enter inside the cell.

The example describes transfection of a gene of interest into a cellusing an embodiment of the conjugate of the present invention, whereinthe gene of interest is mcherry coding for RFP (Red FluorescentProtein). The plasmid containing mcherry gene is linked to a protein toform a conjugate, wherein the conjugate comprises EfsQNR protein andcopper [II] phenanthroline. The conjugate is used to transfect HeLacells. The successful expression of mcherry gene in HeLa cells is shownas a proof-of-concept to establish the ability of the conjugate topenetrate the cell membrane and transfect the cells.

Material and Methods

Copper phenanthroline complex was commercially procured.

Example 6

Protein studies—Studies governing the expression of EfsQNR plasmid,conditions of incubation, and purification were followed as publishedpreviously (Hegde et al. Antimicrob. Agents and Chemother. 2011:55(1):110-7 which is incorporated herein by reference) to obtain EfsQNRprotein. The SDS-PAGE (polyacrylamide gel electrophoresis) was doneusing the Bio-Rad kit and the protein bands were visualised using 12%polyacrylamide gel.

Example 7 Preparation of a Conjugate

As is disclosed in the present invention, a conjugate is prepared fortransfecting cells, the conjugate comprising EfsQNR (SEQ ID NO: 2),copper [II] phenanthroline, and a plasmid having a gene of interest.

FIG. 18 depicts a vector map of plasmid carrying mcherry gene (SEQ IDNO: 25), used in preparation of the conjugate in the present example.EfsQNR protein (SEQ ID NO: 2) is a β helical protein having apentapeptide repeat (as depicted in SEQ ID NO: 18) having length in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm. Copper [II] phenanthroline is a nucleicacid intercalating agent used as a linker in the present example.

EfsQNR protein obtained by governing the expression of EfsQNR plasmidwas complexed with copper [II] phenanthroline complex to obtain acomplex comprising EfsQNR and copper [II] phenanthroline. The obtainedcomplex was further reacted with plasmid having mcherry gene to obtain aconjugate in accordance with an embodiment of the present invention. Theobtained conjugate was used to transfect HeLa cells.

FIG. 19 depicts two schemes for preparing the conjugate that can be usedfor transfection. Scheme 1 suggests the use of copper [II]phenanthroline as a linker which forms a complex with a protein. Theobtained complex is further reacted with a plasmid having a gene ofinterest to obtain the conjugate to be used for transfection.

Scheme 2 suggests the use of a DNA binding protein as a linker whichforms a complex with a protein. The obtained complex is further reactedwith a plasmid having a gene of interest to obtain the conjugate to beused for transfection.

In the present example, copper [II] phenanthroline is used as a linkerto form a complex and a recombinant β helical protein—EfsQNR (SEQ ID NO:2) is used along with a plasmid containing mcherry gene. The followingprotocol was used for preparing the complex and the conjugate:

The protein EfsQNR was mixed with copper [II] phenanthroline in a 1:2molar ratio and incubated for 30 minutes as room temperature to obtainthe complex. Plasmid containing mcherry gene was mixed with the complexobtained in previous step and incubated for 30 minutes at 4° C. toobtain the conjugate. The conjugate so obtained was used to transfectHeLa cells.

Example 8 Transfection of HeLa Cells Using the Conjugate

FIG. 20 depicts a scheme for transfection experiment using the conjugateobtained in Example 9. In the present example following conditions wereused for performing the transfection experiment.

The conjugate comprising the mcherry plasmid as obtained in Example 8was added to HeLa cells (8 hours after seeding). The cells were allowedto grow by incubating at 37° C. and 5% CO₂ conditions for 48 hours.After the incubation, the cells were observed under confocal microscopy.

FIG. 21 depicts confocal microscopy images of HeLa cells after thetransfection experiment. The expression of RFP can be observed aslighter (red) dots inside the HeLa cells thereby, proving the cellpenetration and transfecting ability of the conjugate disclosed in thepresent document.

Although the example as presented herein shows the use of protein EfsQNR(SEQ ID NO: 2) in the preparation of a conjugate for transfection, theproteins having amino acid sequence as depicted in SEQ ID NO: 1 and SEQID NO: 3 to SEQ ID NO: 12 can also be used effectively to form theconjugate. Also, recombinant β helical proteins having a penta-peptiderepeat sequence as depicted in SEQ ID NO: 18 and having a length in therange of 5 nm-25 nm, suitably, 10 nm-15 nm and width is in the range of1 nm-5 nm, suitably 1 nm-3 nm can be used for preparing the conjugate asdescribed in the present disclosure. Similarly, the present exampledepicts the use of copper [II] phenanthroline as a linker but othermolecules like DNA binding protein, metal conjugate, drug metalconjugate, and other nucleic acid intercalating molecule can also beused in the formation of a conjugate to be used effectively as atransfecting agent. One class of a DNA binding protein namely, zincfinger protein having a molecular weight of less than 12 kDa can also beused to form the conjugate. One such zinc finger protein that can beused as a linker has been depicted in SEQ ID NO: 24. In the presentdisclosure, a plasmid (FIG. 18 ) carrying mcherry gene has been used forpreparing the conjugate for transfection and the expression of mcherrygene has been shown as a proof-of-concept. It is contemplated thatessentially any gene of interest for transfection purpose can becomplexed with the complex as disclosed herein to form the conjugate foruse in gene therapy.

Advantages

The present disclosure provides with a conjugate comprising a gene ofinterest which possess the ability of penetrating cell membrane andexpressing the gene of interest. Hence, the conjugate can be used fortransfection and has immense potential to be used in gene therapy. As isknown that delivery of the gene to the cells is a major challenge in thefield of gene therapy, the disclosed conjugate opens new avenue in thisfield. The disclosed conjugate is simple to prepare and can be complexedwith wide variety of genes to be used for gene therapy.

Although particular embodiments of the invention have been disclosedherein in detail, this has been done by way of example and for thepurposes of illustration only. The aforementioned embodiments are notintended to be limiting with respect to the scope of the invention. Itis contemplated by the inventors that various substitutions,alterations, and modifications may be made to the invention withoutdeparting from the spirit and scope of the invention.

TABLE 2 List of sequences SEQ ID Sequences SEQ ID NO: 1MPAKTLESKDYCGESFVSEDRSGQS Protein LESIRFEDCTFRQCNFTEAELNRCKFRECEFVDCNLSLISIPQTSFMEVR FVDCKMLGVNWTSAQWPSVKMEGALSFERCILNDSLFYGLYLAGVKMVEC RIHDANFTEADCEDADFTQSDLKGSTFHNTKLTGASFIDAVNYHIDIFHN DIKRARFSLPEAASLLNSLDIELSD SEQ ID NO: 2GSHMKITYPLPPNLPEQLPLLTNCQ Protein LEDEAILENHLYQQIDLPNQEVRNLVFRDAVFDHLSLANGQFASFDCSNV RFEACDFSNVEWLSGSFHRVTFLRCNLTGTNFADSYLKDCLFEDCKADYA SFRFANFNLVHFNQTRLVESEFFEVTWKKLLLEACDLTESNWLNTSLKGL DFSQNTFERLTFSPNYLSGLKVTPE QAIYLASALGLVITSEQ ID NO: 3 QCTGGADCTSCTGACTGCGNCPNAV Protein TCTNSQHCVKANTCTGSTDCNTAQTCTNSKDCFEANTCTDSTNCYKATAC TNSSGCPGH SEQ ID NO: 4GYSCRAVGVDGRAVTDIQGTCHAKA Protein TGAGAMASGTSEPGSTSTATATGRGATARSTSTGRGTATTTATGTASATS NAIGQGTATTTATGSAGGRATGSATTSSSASQPTQTQTITGPGFQTAKSF ARNTATTTVTASHHHHHH SEQ ID NO: 5DGSCTNTNSQLSANSKCEKSTLTNC Protein YVDKSEVYGTTCTGSRFDGVTITTSTSTGSRISGPGCKISTCIITGGVPA PSAACKISGCTFSAN SEQ ID NO: 6GSHMALALVGEKIDRNRFTGEKIEN Protein STFFNCDFSGADLSGTEFIGCQFYDRESQKGCNFSRAMLKDAIFKSCDLS MADFRNSSALGIEIRHCRAQGADFRGASFMNMITTRTWFCSAYITNTNLS YANFSKVVLEKCELWENRWIGAQVLGATFSGSDLSGGEFSTFDWRAANFT HCDLTNSELGDLDIRGVDLQGVKLD NYQASLLMERLGIAVIGSEQ ID NO: 7 MIDKSAFVHPTAIVEEGASIGANAH Protein IGPFCIVGPHVEIGEGTVLKSHVVVNGHTKIGRDNEIYQFASIGEVNQDL KYAGEPTRVEIGDRNRIRESVTIHRGTVQGGGLTKVGSDNLLMINAHIAH DCTVGNRCILANNATLAGHVSVDDFAIIGGMTAVHQFCIIGAHVMVGGCS GVAQDVPPYVIAQGNHATPFGVNIEGLKRRGFSREAITAIRNAYKLIYRS GKTLDEVKPEIAELAETYPEVKAFT DFFARSTRGLIRSEQ ID NO: 8 MGSSHHHHHHSSGLVPRGSHMDVEK Protein LRQLYAAGERDFSIVDLRGAVLENINLSGAILHGAMLDEANLQQANLSRA DLSGATLNGADLRGANLSKADLSDAILDNAILEGAILDEAVLNQANLKAA NLEQAILSHANIREADLSEANLEAADLSGADLAIADLHQANLHQAALERA NLTGANLEDANLEGTILEGGNNNLA T SEQ ID NO: 9ATDTGGYAATAGGNVTGAVSKTATS Protein MQDIVNIIDAARLDANGKKVKGGAYPLVITYTGNEDSLINAAAANICGQW SKDPRGVEIKEFTKGITIIGANGSSANFGIWIKKSSDVVVQNMRIGYLPG GAKDGDMIRVDDSPNVWVDHNELFAANHECDGTPDNDTTFESAVDIKGAS NTVTVSYNYIHGVKKVGLDGSSSSDTGRNITYHHNYYNDVNARLPLQRGG LVHAYNNLYTNITGSGLNVRQNGQALIENNWFEKAINPVTSRYDGKNFGT WVLKGNNITKPADFSTYSITWTADTKPYVNADSWTSTGTFPTVAYNYSPV SAQCVKDKLPGYAGVGKNLATLTST ACK SEQ ID NO: 10VGTNTGGVLVITDTIIVKSGQTYDG Protein KGIKIIAQGMGDGSQSENQKPIFKLEKGANLKNVIIGAPGCDGIHCYGDN VVENVVWEDVGEDALTVKSEGVVEVIGGSAKEAADKVFQLNAPCTFKVKN FTATNIGKLVRQNGNTTFKVVIYLEDVTLNNVKSCVAKSDSPVSELWYHN LNVNNCKTLFEFPSQSQIHQY SEQ ID NO: 11QEITVDEFSNIRENPVTPWNPEPSA Protein PVIDPTAYIDPQASVIGEVTIGANVMVSPMASIRSDEGMPIFVGDRSNVQ DGVVLHALETINEEGEPIEDNIVEVDGKEYAVYIGNNVSLAHQSQVHGPA AVGDDTFIGMQAFVFKSKVGNNCVLEPRSAAIGVTIPDGRYIPAGMVVTS QAEADKLPEVTDDYAYSHTNEAVVY VNVHLAEGYKETSSEQ ID NO: 12 VGVSGSAEGFAKGVTGGGSATPVYP ProteinDTIDELVSYLGDDEARVIVLTKTFD FTDSEGTTTGTGCAPWGTASACQVAIDQDDWCENYEPDAPSVSVEYYNAG TLGITVTSNKSLIGEGSSGAIKGKGLRIVSGAENIIIQNIAVTDINPKYV WGGDAITLDDCDLVWIDHVTTARIGRQHYVLGTSADNRVSLTNNYIDGVS DYSATCDGYHYWAIYLDGDADLVTMKGNYIYHTSGRSPKVQDNTLLHAVN NYWYDISGHAFEIGEGGYVLAEGNVFQNVDTVLETYEGEAFTVPSSTAGE VCSTYLGRDCVINGFGSSGTFSEDSTSFLSDFEGKNIASASAYTSVASRV VANAGQGNL SEQ ID NO: 13AYTLATHTAGVIPAGKLERVDPTTV Protein RQEGPWADPAQAVVQTGPNQYTVYVLAFAFGYQPNPIEVPQGAEIVFKIT SPDVIHGFHVEGTNINVEVLPGEVSTVRYTFKRPGEYRIICNQYCGLGHQ NMFGTIVVKE SEQ ID NO: 14 PAAKRVKCD PeptideSEQ ID NO: 15 YPYDVPDYAKDEL Peptide SEQ ID NO: 16MLSLRQSIRFFKPATRTLCSSRYLL Peptide SEQ ID NO: 17LSTAADMQGVVTDGMASGLDKDYLK Peptide PDD SEQ ID NO: 18(STAV)₁(DN)₂(LF)₃(STR)₄(G)₅ consensus sequence SEQ ID NO: 19ATGCCGGCGAAAACCCTGGAAAGCA DNA AAGATTATTGCGGCGAAAGCTTTGTGAGCGAAGATCGCAGCGGCCAGAGC CTGGAAAGCATTCGCTTTGAAGATTGCACCTTTCGCCAGTGCAACTTTAC CGAAGCGGAACTGAACCGCTGCAAATTTCGCGAATGCGAATTTGTGGATT GCAACCTGAGCCTGATTAGCATTCCGCAGACCAGCTTTATGGAAGTGCGC TTTGTGGATTGCAAAATGCTGGGCGTGAACTGGACCAGCGCGCAGGCGGG CGCGCTGAGCTTTGAACGCTGCATTCTGAACGATAGCCTGTTTTATGGCC TGTATCTGGCGGGCGTGAAAATGGTGGAATGCCGCATTCATGATGCGAAC TTTACCGAAGCGGATTGCGAAGATGCGGATTTTACCCAGAGCGATCTGAA AGGCAGCACCTTTCATAACACCAAACTGACCGGCGCGAGCTTTATTGATG CGGTGAACTATCATATTGATATTTTTCATAACGATATTAAACGCGCGCGC TTTAGCCTGCCGGAAGCGGCGAGCCTGCTGAACAGCCTGGATATTGAACT GAGCGAT SEQ ID NO: 20GGCAGCCATATGAAAATTACCTATC DNA CGCTGCCGCCGAACCTGCCGGAACAGCTGCCGCTGCTGACCAACTGCCAG CTGGAAGATGAAGCGATTCTGGAAAACCATCTGTATCAGCAGATTGATCT GCCGAACCAGGAAGTGCGCAACCTGGTGTTTCGCGATGCGGTGTTTGATC ATCTGAGCCTGGCGAACGGCCAGTTTGCGAGCTTTGATTGCAGCAACGTG CGCTTTGAAGCGTGCGATTTTAGCAACGTGGAATGGCTGAGCGGCAGCTT TCATCGCGTGACCTTTCTGCGCTGCAACCTGACCGGCACCAACTTTGCGG ATAGCTATCTGAAAGATTGCCTGTTTGAAGATTGCAAAGCGGATTATGCG AGCTTTCGCTTTGCGAACTTTAACCTGGTGCATTTTAACCAGACCCGCCT GGTGGAAAGCGAATTTTTTGAAGTGACCTGGAAAAAACTGCTGCTGGAAG CGTGCGATCTGACCGAAAGCAACTGGCTGAACACCAGCCTGAAAGGCCTG GATTTTAGCCAGAACACCTTTGAACGCCTGACCTTTAGCCCGAACTATCT GAGCGGCCTGAAAGTGACCCCGGAACAGGCGATTTATCTGGCGAGCGCGC TGGGCCTGGTGATTACC SEQ ID NO: 21GCGTATACCCTGGCGACCCATACCG DNA CGGGCGTGATTCCGGCGGGCAAACTGGAACGCGTGGATCCGACCACCGTG CGCCAGGAAGGCCCGTGGGCGGATCCGGCGCAGGCGGTGGTGCAGACCGG CCCGAACCAGTATACCGTGTATGTGCTGGCGTTTGCGTTTGGCTATCAGC CGAACCCGATTGAAGTGCCGCAGGGCGCGGAAATTGTGTTTAAAATTACC AGCCCGGATGTGATTCATGGCTTTCATGTGGAAGGCACCAACATTAACGT GGAAGTGCTGCCGGGCGAAGTGAGCACCGTGCGCTATACCTTTAAACGCC CGGGCGAATATCGCATT ATTTGCAACCAGTATTGCGGCCTGGGCCATCAGAACATGTTTGGCACCAT TGTGGTGAAAGAA SEQ ID NO: 22 GDVQKKRWLFETKPLDpeptide SEQ ID NO: 23 VQSKCGSKDNIKHVPGGG peptide SEQ ID NO: 24MERPYACPVESCDRRFSDSSNLTRH peptide IRIHTGQKPFQCRICMRNFSRSDHLTTHIRTHTGEKPFACDICGRKFARS DERKRHTKIHLRQKD SEQ ID NO: 25GTGAGCAAGGGCGAGGAGGATAACA peptide TGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCC GTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTA CGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGC CCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCC TACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCC CGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGG TGACCGTGACCCAGGACTCCTCCCTCCAGGACGGCGAGTTCATCTACAAG GTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAA GAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACG GCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGC CACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCA GCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACA ACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCAC TCCACCGGCGGCATGGACGAGCTGTACAAGTAGTAATCTAGAGGGCCCTA TTCTATAGTGTCACC SEQ ID NO: 26ATCCCGCTCATATGCCGGCCAAGAC DNA CCTTG SEQ ID NO: 27ATCCCGCTCTCGAGTCAATCGGACA DNA GCTCGATATC SEQ ID NO: 28ATCCCGCTCATATGAAAATAACTTA DNA TCCCTTGCCA SEQ ID NO: 29ATCCCGCTCTCGAGTTAGGTAATCA DNA CCAAACCAAGT 

1. A cell penetrating conjugate comprising a recombinant β helicalprotein linked to a functional molecule, wherein the β helical proteinlength is in the range of from 5 nm to 25 nm, and width is in the rangeof from 1 nm to 5 nm.
 2. The conjugate as claimed in claim 1, whereinthe β helical protein length is in the range of from 10 nm to 15 nm, andwidth is in the range of from 1 nm to 3 nm.
 3. The conjugate as claimedin claim 1, wherein the β helical protein comprises one or more aminoacid ladder structures selected from the group consisting of an arginineladder; a lysine ladder; an asparagine ladder; an aspartic acid ladder;and a glutamic acid ladder.
 4. The conjugate as claimed in claim 3,wherein when present, the arginine ladder comprises from 10 to 20arginine residues; the lysine ladder comprises from 10 to 30 lysineresidues; the asparagine ladder comprises from 10 to 40 asparagineresidues; the aspartic acid ladder comprises from 10 to 40 aspartic acidresidues; and the glutamic acid ladder comprises from 10 to 40 glutamicacid residues.
 5. The conjugate as claimed in claim 1, wherein the βhelical protein has a total charge that is less than zero.
 6. Theconjugate as claimed in claim 5, wherein the β helical protein has atotal charge of from −20 to −60.
 7. The conjugate as claimed in claim 1,wherein the β helical protein has a β helical structure with a stiffnessparameter K (beta helix) 0.2 to 12 N/m², as measured by atomic forcemicroscopy.
 8. The conjugate as claimed in claim 1, wherein the βhelical protein is a pentapeptide-repeat protein.
 9. The conjugate asclaimed in claim 8, wherein the β helical protein comprises tandemlyrepeated pentapeptide with consensus sequence(STAV)₁(DN)₂(LF)₃(STR)₄(G)₅.
 10. The cell penetrating conjugate asclaimed in claim 1, wherein the β helical protein is represented by asequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,and combinations thereof.
 11. The cell penetrating conjugate as claimedin claim 1, wherein the recombinant β helical protein is linked to thefunctional molecule via a linker molecule selected from the groupconsisting of: polyethyleneglycol (PEG); peptide; metal conjugate,drug-metal conjugate, DNA binding domain, nucleic acid intercalatingmolecule and combinations thereof.
 12. The cell penetrating conjugate asclaimed in claim 11, wherein when the linker molecule is a peptide, thepeptide comprises amino acids selected from the group consisting of:aliphatic amino acids; aromatic amino acids; and combinations thereof.13. The cell penetrating conjugate as claimed in claim 1, wherein thelinker and the recombinant β helical protein are linked by one or moreof the group selected consisting of: covalent bonds; non-covalent bonds;and combinations thereof.
 14. The cell penetrating conjugate as claimedin claim 1, wherein the recombinant β helical protein is linked to afunctional molecule via by a linkage selected from the group consistingof: an ester linkage; and an amide linkage.
 15. The cell penetratingconjugate as claimed in claim 1, wherein the functional molecule isselected from the group consisting of dyes, drugs, metal, drug-metalconjugate, proteins, enzymes, antibodies, nucleic acids,polysaccharides, nuclear localizing signals, nanoparticles, andcombinations thereof.
 16. The cell penetrating conjugate as claimed inclaim 1, wherein the conjugate further comprises a signal sequencewherein the signal sequence directs the conjugate to a particular cellor part of a cell.
 17. The cell penetrating conjugate as claimed inclaim 9, wherein the signal sequence is selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ IDNO:
 17. 18. The cell penetrating conjugate as claimed in claim 1,wherein the conjugate further comprises a phosphatidyl choline molecule.19. The cell penetrating conjugate as claimed in claim 9, wherein theconjugate transfers the functional molecule to a location selected fromthe group consisting of: cell organelles; nucleus; endoplasmicreticulum; mitochondria; cell membrane; and P-cadherin overexpressingbreast cancer cells.
 20. The cell penetrating conjugate as claimed inclaim 12, wherein the cell organelles are selected from the groupcomprising actin filaments, golgi bodies, cell membrane, microtubulins.21. A process for transferring a functional molecule into a cell, saidprocess comprising: a) linking the functional molecule to a recombinantβ helical protein to obtain a conjugate; b) contacting the conjugatewith at least one cell; wherein contacting the conjugate of step (b)transfers the functional molecule into the cell; and wherein the βhelical protein length is in the range of from 5 nm to 25 nm and widthis in the range of from 1 nm to 5 nm.
 22. The process as claimed inclaim 21, wherein the process comprises after step (b): c) detecting thetransfer of the conjugate inside the cell.
 23. The process as claimed inclaim 21, wherein the cell is selected from the group consisting ofeukaryotic cells, prokaryotic cells, and combinations thereof.